PCR reaction vessel, PCR device, PCR method

Abstract

Provided are a PCR reaction container, a PCR device, and a PCR method. The PCR reaction container (10) includes: a substrate (14); a flow path (12) formed in the substrate (14); a pair of a first filter (28) and a second filter (30) provided at both ends of the flow path (12); a pair of a first air communication port (24) and a second air communication port (26) that communicate with the flow path (12) through the first filter (28) and the second filter (30); a thermal cycle region (12e) formed between the first filter (28) and the second filter (30) in the flow path (12); a branching point (112c) formed between the first filter (28) and the second filter (30) in the flow path (12); a branched flow path (131) having one end connected to the branching point (112c); and a sample introduction port (133) formed at the other end of the branched flow path (131).

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201680069395.8 (international application No. PCT / JP2016 / 085216), with the Chinese national phase entry date of May 28, 2018 (international application date of November 28, 2016) and the invention title "PCR reaction vessel, PCR apparatus, PCR method". Technical Field

[0002] This invention relates to PCR reaction containers for polymerase chain reaction (PCR) and PCR apparatus and PCR methods using such PCR reaction containers. Background Technology

[0003] Genetic testing is widely used not only in various medical fields for detection, identification of crops or pathogenic microorganisms, and evaluation of food safety, but also for the detection of pathogenic viruses and various infectious diseases. Methods for analyzing samples obtained by amplifying a portion of DNA to detect trace amounts of genetic material with high sensitivity are known. In particular, PCR (polymerase chain reaction) is a prominent technique for selectively amplifying a specific portion of DNA extracted from living organisms. PCR involves subjecting a live sample containing DNA and a mixture of PCR reagents (primers or enzymes) to a predetermined thermal cycle, causing repeated denaturation, annealing, and extension reactions to selectively amplify specific portions of the DNA.

[0004] In PCR, a predetermined number of samples are typically placed into PCR tubes or microplates with multiple wells. However, in recent years, the use of reaction vessels with microchannels (also called chips) formed on a substrate has become practical. Regardless of the type of reaction vessel, various technological advancements have enabled the application of predetermined thermal cycling at high speed and precision within the vessel.

[0005] Patent Document 1 discloses a reaction vessel having a flow channel for performing PCR. In this reaction vessel, a flow channel is formed between two overlapping resin substrates, and a sample inlet for introducing a sample into the flow channel through a through-hole 1 formed on the resin substrate, and a sample outlet for discharging the sample. A temperature control section, such as a Peltier element, is disposed in a recessed area on the back of the resin substrate. A nozzle is disposed at the sample inlet of the reaction vessel, and the sample can be moved in the flow channel by supplying and drawing air through the nozzle.

[0006] [Prior Technology Documents]

[0007] [Patent Literature]

[0008] Patent Document 1: Japanese Patent Application Publication No. 2009-232700 Summary of the Invention

[0009] [The problem the invention aims to solve]

[0010] In PCR, it is crucial to prevent contamination from external sources into the system during sample preparation. If contamination includes live specimens other than the target sample, it may amplify the DNA contained in those specimens. In this case, the target sample cannot be used for accurate subsequent analysis. Therefore, methods must be devised to prevent contamination after the sample is introduced into the reaction vessel.

[0011] However, in the invention disclosed in Patent Document 1, for example, if a live specimen other than the target being processed is attached to the nozzle or the pump supplying air to the nozzle, the live specimen may enter the reaction vessel through the sample inlet, resulting in contamination. Furthermore, from both cost and environmental perspectives, it is impractical to replace the nozzle, pump front-end parts, and accessories for each PCR process.

[0012] The present invention was developed in view of the following situation, and its object is to provide a PCR reaction container that can properly prevent contamination, a PCR apparatus using the PCR reaction container, and a PCR method.

[0013] [Methods used to solve problems]

[0014] To solve the above problems, one embodiment of the present invention provides a PCR reaction vessel comprising: a substrate; a flow channel formed on the substrate; a pair of filters disposed at both ends of the flow channel; a pair of air inlets connected to the flow channel through the filters; a thermal circulation region formed between the pair of filters in the flow channel; a bifurcation point formed between the pair of filters in the flow channel; a bifurcation flow channel, one end of which is connected to the bifurcation point; and a sample inlet formed at the other end of the bifurcation flow channel.

[0015] Another embodiment of the present invention is also a PCR reaction vessel. This PCR reaction vessel has: a substrate; a flow channel formed on the substrate; a pair of filters disposed at both ends of the flow channel; a pair of air inlets connected to the flow channel via the filters; a thermal cycling region formed between the pair of filters in the flow channel; a first branch point formed between the pair of filters in the flow channel; a first branch flow channel, one end of which is connected to the first branch point; a first sample inlet formed at the other end of the first branch flow channel; a second branch point formed between the pair of filters in the flow channel; a second branch flow channel, one end of which is connected to the second branch point; and a second sample inlet formed at the other end of the second branch flow channel.

[0016] The PCR reaction vessel described above may also have a buffer channel region formed between the first and second bifurcation points in the channel.

[0017] The buffer channel area can be set to a predetermined volume corresponding to the amount of sample to be subjected to PCR treatment.

[0018] The thermal circulation region may include serpentine flow channels. The thermal circulation region may also include: a pair of reaction regions each containing serpentine flow channels; and a connecting region for connecting the pair of reaction regions.

[0019] It can also have a sealing film for sealing the air vent and sample inlet.

[0020] The sealing film can be formed so that it can be pierced by a needle.

[0021] Another embodiment of the present invention is a PCR apparatus. This apparatus may include: the aforementioned PCR reaction vessel; a temperature control unit for regulating the temperature of the thermal cycling zone; and a pump system for controlling the pressure within the flow channel via an air vent to move the sample within the thermal cycling zone.

[0022] The pump system can be a type of air pump where the pressure on the primary and secondary sides becomes equal when the pump stops.

[0023] The air pump may have a nozzle with a hollow needle at the front end.

[0024] The PCR device may also have a fluorescence detector for detecting fluorescence emitted from the sample in the flow channel.

[0025] The PCR device may also have a control unit that controls the pump system based on the values ​​detected by the fluorescence detector.

[0026] Another embodiment of the present invention is a PCR method. The method includes: a step of preparing a PCR reaction container; a step of introducing a sample into the PCR reaction container via a sample inlet; a step of setting the PCR reaction container in a PCR apparatus having a pump; a step of connecting the pump nozzle to an air vent; and a step of moving the sample within a thermal cycling region by controlling the pressure within the flow channel by the pump. The PCR reaction container includes: a substrate; a flow channel formed on the substrate; a pair of filters disposed at both ends of the flow channel; a pair of air vents connected to the flow channel via the filters; a thermal cycling region formed between the pair of filters in the flow channel; a bifurcation point formed between the pair of filters in the flow channel; a bifurcation flow channel, one end of which is connected to the bifurcation point; and a sample inlet formed at the other end of the bifurcation flow channel.

[0027] In the step of moving the sample, the branched channels can retain samples that are not used for PCR.

[0028] Another embodiment of the present invention is also a PCR method. This PCR method includes: a step of preparing a PCR reaction container; a step of introducing a sample into the PCR reaction container through a first sample inlet or a second sample inlet; a step of setting the PCR reaction container in a PCR apparatus having a pump; a step of connecting the pump nozzle to an air vent; and a step of moving the sample within a thermal cycling region by controlling the pressure within the flow channel by the pump. The PCR reaction container includes: a substrate; a flow channel formed on the substrate; a pair of filters disposed at both ends of the flow channel; a pair of air vents connected to the flow channel through the filters; a thermal cycling region formed between the pair of filters in the flow channel; a first branch point formed between the pair of filters in the flow channel; a first branch flow channel, one end of which is connected to the first branch point; a first sample inlet formed at the other end of the first branch flow channel; a second branch point formed between the pair of filters in the flow channel; a second branch flow channel, one end of which is connected to the second branch point; and a second sample inlet formed at the other end of the second branch flow channel.

[0029] The PCR reaction vessel may also have a buffer channel region formed in the channel between the first and second bifurcation points, and the PCR method described above may also have a step of using the buffer channel region to dispense samples.

[0030] During the sample movement step, samples not intended for PCR may remain in the first and second branch channels.

[0031] [Invention Effects]

[0032] According to the present invention, a PCR reaction container capable of appropriately preventing contamination, a PCR apparatus using the PCR reaction container, and a PCR method can be provided. Attached Figure Description

[0033] Figure 1 (a) and (b) are diagrams illustrating the PCR reaction vessel according to the first embodiment of the present invention.

[0034] Figure 2 yes Figure 1 (a) shows an AA cross-section of the PCR reaction vessel.

[0035] Figure 3 yes Figure 1 (a) shows a BB cross-section of the PCR reaction vessel.

[0036] Figure 4 This is a top view of the substrate of the PCR reaction vessel of the first embodiment.

[0037] Figure 5 This is a schematic diagram illustrating the structure of the PCR reaction vessel according to the first embodiment.

[0038] Figure 6 This is a schematic diagram illustrating the situation when the sample is introduced into the PCR reaction vessel in the first embodiment.

[0039] Figure 7 This diagram shows the state when the third sealing film is pasted back onto the substrate in the first embodiment.

[0040] Figure 8 This is a diagram illustrating a PCR apparatus that uses the PCR reaction vessel described in the first embodiment.

[0041] Figure 9 This is a diagram illustrating the state when the PCR reaction vessel is positioned at a predetermined location in the PCR apparatus in the first embodiment.

[0042] Figure 10 This diagram illustrates the situation in the first embodiment where the nozzle of the pump system is connected to the air vent of the PCR reaction vessel.

[0043] Figure 11 yes Figure 10 The diagram shows a cross-sectional view of the PCR reaction vessel.

[0044] Figure 12 This is a diagram illustrating the situation in the first embodiment where the pump system is operated to move the sample.

[0045] Figure 13 (a) and (b) are diagrams illustrating the PCR reaction vessel according to the second embodiment of the present invention.

[0046] Figure 14 yes Figure 13 (a) shows an AA cross-section of the PCR reaction vessel.

[0047] Figure 15 yes Figure 13 (a) shows a BB cross-section of the PCR reaction vessel.

[0048] Figure 16 This is a top view of the substrate of the PCR reaction vessel of the second embodiment.

[0049] Figure 17 This is a schematic diagram illustrating the structure of the PCR reaction vessel involved in the second embodiment.

[0050] Figure 18 This is a schematic diagram illustrating the process of introducing a sample into a PCR reaction vessel in the second embodiment.

[0051] Figure 19 This diagram shows the state when the third sealing film is pasted back onto the substrate in the second embodiment.

[0052] Figure 20 This is a diagram illustrating a PCR apparatus that uses the PCR reaction vessel described in the second embodiment.

[0053] Figure 21 This is a diagram illustrating the state when the PCR reaction vessel is positioned at a predetermined location in the PCR apparatus in the second embodiment.

[0054] Figure 22 This diagram illustrates the second embodiment, where the nozzle of the pump system is connected to the air vent of the PCR reaction vessel.

[0055] Figure 23 yes Figure 22 The diagram shows a cross-sectional view of the PCR reaction vessel.

[0056] Figure 24 This is a diagram illustrating the situation in the second embodiment where the pump system is operated to move the sample. Detailed Implementation

[0057] The PCR reaction vessel and PCR apparatus according to embodiments of the present invention will now be described. Identical or equivalent components, parts, and processing reference numerals are used in the accompanying drawings, and repeated descriptions are omitted where appropriate. Furthermore, these embodiments are not intended to limit the invention, but are illustrative; not all features or combinations thereof described in the embodiments are essential elements of the present invention.

[0058] [First Implementation]

[0059] Figure 1 (a) and (b) are diagrams illustrating the PCR reaction vessel 10 according to the first embodiment of the present invention. Figure 1 (a) is a top view of PCR reaction vessel 10. Figure 1 (b) is a front view of PCR reaction vessel 10. Figure 2 yes Figure 1 (a) shows a cross-sectional view of the PCR reaction vessel 10. Figure 3 yes Figure 1 (a) shows a BB cross-section of the PCR reaction vessel 10. Figure 4 This is a top view of the substrate 14 of the PCR reaction vessel 10. Figure 5 This is a schematic diagram illustrating the structure of the PCR reaction vessel 10.

[0060] The PCR reaction vessel 10 is composed of the following elements: a resin substrate 14, on which a groove-shaped flow channel 12 is formed; a flow channel sealing film 16, which is attached to the bottom surface 14a of the substrate 14 and is used to seal the flow channel 12; and three sealing films (a first sealing film 18, a second sealing film 20 and a third sealing film 22), which are attached to the top surface 14b of the substrate 14.

[0061] The substrate 14 is preferably formed of a material with good thermal conductivity, good stability relative to temperature changes, and low permeability to the sample solution used. Furthermore, the substrate 14 is preferably formed of a material with high plasticity, good transparency and insulation, and weak autofluorescence. Such materials are preferably inorganic materials such as glass and silicon, or resins such as acrylic resin, polyester, and silicone, with cycloolefins being particularly preferred. As an example of the dimensions of the substrate 14, the long side is 70 mm, the short side is 42 mm, and the thickness is 3 mm. As an example of the dimensions of the flow channel 12 formed on the bottom surface 14a of the substrate 14, the width is 0.5 mm and the depth is 0.5 mm.

[0062] As described above, a groove-shaped flow channel 12 is formed on the bottom surface 14a of the substrate 14, and the flow channel 12 is sealed by a flow channel sealing film 16 (see reference). Figure 2 In the substrate 14, a first air vent 24 is formed at one end 12a of the flow channel 12. In the substrate 14, a second air vent 26 is formed at the other end 12b of the flow channel 12. The pair of first air vents 24 and second air vents 26 are formed to protrude from the top surface 14b of the substrate 14. Such a substrate can be manufactured by injection molding or by machining using an NC machining machine or the like.

[0063] In the substrate 14, a first filter 28 is provided between the first air vent 24 and one end 12a of the flow channel 12 (see reference). Figure 2 In the substrate 14, a second filter 30 is provided between the second air vent 26 and the other end 12b of the flow channel 12. The pair of first filters 28 and second filters 30 provided at both ends of the flow channel 12 have good low-impurity characteristics and allow only air to pass through, thereby preventing contamination and ensuring that the quality of the DNA amplified by PCR does not deteriorate. As the filter material, polyethylene or PTFE is preferred, and it may also be porous or hydrophobic. The dimensions of the first filter 28 and the second filter 30 are formed so that they can be precisely accommodated in the filter placement space formed on the substrate 14.

[0064] In substrate 14, a branched flow channel 131 branching off from flow channel 12 is formed at the bifurcation point 112c between the first filter 28 and the second filter 30. A sample inlet 133 (see reference 14) is formed at the end 31a of the branched flow channel 131. Figure 3 The sample inlet 133 is formed to be exposed on the top surface 14b of the substrate 14.

[0065] In the flow channel 12, the portion between the first filter 28 and the bifurcation point 112c forms a thermal circulation region 12e for applying thermal circulation to the sample. This thermal circulation region 12e is pre-defined to include a high-temperature region and a medium-temperature region. The thermal circulation region 12e of the flow channel 12 includes a serpentine flow path. This is to efficiently transfer the heat provided by the PCR device during the PCR process to the sample, and to ensure that the volume of sample available for PCR is sufficient. Although a bifurcation point 112c is provided between the thermal circulation region 12e and the second filter 30 in this first embodiment, since the bifurcation point 112c is used to introduce the sample for PCR into the flow channel through the bifurcation flow path 131 connected to it and the sample inlet 133, its function is not problematic as long as it is formed between the first filter 28 and the second filter 30. Since the PCR reaction vessel 10 is placed in the PCR apparatus and is intended to thermally cycle the sample and measure optical properties such as fluorescence emitted from the sample, the configuration of each element, including the flow channel and the bifurcation point, can be arbitrarily selected, taking into account the configuration of the temperature control unit and the fluorescence detection detector described below. In this first embodiment, the bifurcation point 112c is positioned closer to the second filter 30, and the thermal cycling region is designed between the bifurcation point 112c and the first filter 28. This allows for a larger distance in the flow channel between the bifurcation point 112c and the first filter 28, providing space for an efficient configuration of the thermal cycling region and for the temperature control unit when placed in the PCR apparatus. Conversely, when the bifurcation point 112c is positioned closer to the first filter 28, it is more reasonable to form a thermal cycling region 12e between the bifurcation point 112c and the second filter 30.

[0066] In the PCR reaction vessel 10 according to this first embodiment, most of the flow channel 12 is formed as a groove exposed on the bottom surface 14a of the substrate 14. This is so that it can be easily formed by injection molding using a mold or the like. To utilize this groove as a flow channel, a flow channel sealing film 16 is attached to the bottom surface 14a of the substrate 14. The flow channel sealing film 16 may have adhesiveness on one of its main surfaces, or it may have a functional layer formed on one of its main surfaces that can exert adhesiveness or bonding by pressing, thereby having the function of easily adhering to the bottom surface 14a of the substrate 14 and becoming integrated. The flow channel sealing film 16 is preferably formed of a material with weak autofluorescence, including the adhesive. In this regard, a transparent film made of resins such as cyclic olefin polymers, polyesters, polypropylene, polyethylene, or acrylic resins is more suitable, but not limited to this. Alternatively, the flow channel sealing film 16 may also be formed of plate-shaped glass or resin. At this point, because it possesses rigidity, it can effectively prevent the PCR reaction container 10 from warping or deforming.

[0067] Furthermore, in the PCR reaction vessel 10 according to this first embodiment, the first air vent 24, the second air vent 26, the first filter 28, the second filter 30, and the sample inlet 133 are all exposed on the top surface 14b of the substrate 14. Therefore, a first sealing film 18 is adhered to the top surface 14b of the substrate 14 to seal the first air vent 24 and the first filter 28. Furthermore, a second sealing film 20 is adhered to the top surface 14b of the substrate 14 to seal the second air vent 26 and the second filter 30. Furthermore, a third sealing film 22 is adhered to the top surface 14b of the substrate 14 to seal the sample inlet 133.

[0068] The first sealing film 18 is a sealing film capable of simultaneously sealing the size of the first air vent 24 and the first filter 28, and the second sealing film 20 is a sealing film capable of simultaneously sealing the size of the second air vent 26 and the second filter 30. The booster pump (described below) is connected to the first air vent 24 and the second air vent 26 by a hollow needle (a sharp-tipped injection needle) provided at the front end of the pump, which is inserted into the first air vent 24 and the second air vent 26. Therefore, the first sealing film 18 and the second sealing film 20 are preferably films formed of a material and thickness that are easily perforated by a needle. This first embodiment describes a sealing film capable of simultaneously sealing the size of the corresponding air vent and filter, but it is also possible to seal them separately. Alternatively, a sealing film capable of sealing the first air vent 24, the first filter 28, the second air vent 26, and the second filter 30 together (on a single sheet) is also possible.

[0069] The third sealing film 22 is a sealing film capable of sealing the sample inlet 133. The sample is introduced into the flow channel 12 through the sample inlet 133 by temporarily peeling the third sealing film 22 off the substrate 14. After a predetermined amount of sample is introduced, the third sealing film 22 is then adhered back to the top surface 14b of the substrate 14. Therefore, the third sealing film 22 is preferably a film with adhesiveness capable of withstanding multiple rounds of adhesion and peeling. Furthermore, the third sealing film 22 can also be a new film adhered after the sample is introduced; in this case, the importance of adhesion and peeling characteristics can be reduced.

[0070] Furthermore, when introducing the sample, either the first sealing film 18 or the second sealing film 20 needs to be temporarily peeled off, similar to the third sealing film 22. This is because the sample will not enter the flow channel if an air outlet is not provided. Therefore, the first sealing film 18 and the second sealing film 20 are preferably films with the same adhesiveness capable of withstanding multiple rounds of application and peeling. Alternatively, a new film can be applied after the sample has been introduced.

[0071] The first sealing film 18, the second sealing film 20, and the third sealing film 22, like the flow channel sealing film 16, may have an adhesive layer formed on one of their main surfaces, or a functional layer capable of exerting adhesion or bonding through pressing. Preferably, the first sealing film 18, the second sealing film 20, and the third sealing film 22, including the adhesive, are formed of a material with weak self-fluorescence. In this regard, transparent films made of resins such as cycloolefins, polyesters, polypropylene, polyethylene, or acrylic resins are suitable, but not limited to these. Furthermore, it is preferable that, as described above, their adhesive properties do not deteriorate to the point of affecting their use even after repeated application and peeling. However, in cases where a new film is applied after peeling and introducing a sample, the importance of these adhesion and peeling properties can be reduced.

[0072] Next, the method of using the PCR reaction vessel 10 constructed as described above will be explained. First, prepare a sample to be amplified by thermal cycling. Examples of samples include products obtained by adding various primers, thermostable enzymes, and four deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP) as PCR reagents to a mixture containing two or more DNAs. Next, peel off the first sealing film 18 and the third sealing film 22 from the substrate 14 to open the first air vent 24 and the sample inlet 133. When the first sealing film 18 is large enough to simultaneously seal the first air vent 24 and the first filter 28, the first sealing film 18 can be completely peeled off the substrate 14 to open the first air vent 24 and the first filter 28 to the atmosphere. However, by not completely peeling the first sealing film 18 off the substrate 14, but only opening the first air vent 24, the first filter 28 is not exposed to the atmosphere, effectively preventing contamination. Furthermore, the same applies when using a sealing membrane that can seal the first air vent 24 and the first filter 28 respectively, so that the first filter 28 is not exposed to the atmosphere, and contamination can be effectively prevented.

[0073] Next, use a pipette or syringe to introduce the sample into the sample inlet 133. Figure 6 The illustration schematically shows the process of introducing sample 70 into PCR reaction vessel 10. Furthermore, Figure 6 To highlight the position of sample 70, a solid line thicker than flow channel 12 is used to represent sample 70. Please note that this does not indicate that sample 70 is overflowing from the flow channel.

[0074] like Figure 6 As shown, the sample 70 introduced into the sample inlet 133 is forced in by a liquid suction tube or syringe, or filled into the flow channel by capillary action. The sample 70 is filled beyond the bifurcation point 112c in the flow channel 12 and moves towards the thermal cycling region 12e (in the direction of the first air connection 24). However, the sample 70 does not fill beyond the bifurcation point 112c and move towards the second air connection 26. This is because the second air connection 26 is sealed and has no air outlet.

[0075] Next, as Figure 7 As shown, the first sealing film 18 and the third sealing film 22 are then adhered back to the substrate 14 to seal the first air vent 24 and the sample inlet 133. Alternatively, new first sealing films 18 and third sealing films 22 can be adhered as described above. This completes the process of introducing sample 70 into the PCR reaction vessel 10.

[0076] Figure 8This is a diagram illustrating a PCR apparatus 100 that uses a PCR reaction vessel 10. Figure 9 This is a diagram illustrating the state when the PCR reaction vessel 10 is set in the predetermined position of the PCR apparatus 100.

[0077] The PCR apparatus 100 includes a photodetector 122 for fluorescence detection, a first heater 134, and a second heater 135. For example... Figure 9 As shown, the PCR reaction vessel 10 is arranged in the PCR apparatus 100 as follows: the two reaction regions of the thermal cycling region 12e of the flow channel 12 are respectively arranged on the first heater 134 and the second heater 135, and the fluorescence detection optical detector 122 is arranged in the connection region between the two reaction regions.

[0078] The PCR apparatus 100 also includes a pump system 110 for reciprocating motion of the sample 70 within the thermal cycling zone 12e. This pump system 110 includes a first nozzle 101, a second nozzle 102, a first pump 103, a second pump 104, a first actuator 105, a second actuator 106, and a control unit 107. The first nozzle 101 of the pump system 110 is connected to the first air vent 24 of the PCR reaction vessel 10, and the second nozzle 102 of the pump system 110 is connected to the second air vent 26 of the PCR reaction vessel 10. The specific connection method between the nozzles and the air vents will be described below. The pump system 110 controls the pressure within the flow channel 12 via the first air vent 24 and the second air vent 26 to move the sample within the thermal cycling zone 12e.

[0079] In the PCR apparatus 100 according to this first embodiment, the first heater 134 and the second heater 135 are set to different temperatures. Each heater is a component that supplies heat to control the temperature of two reaction regions in the thermal circulation region 12e, and has an area capable of covering the area of ​​each reaction region. Furthermore, each heater can be a resistance heater or a Peltier element, or similar means or construction. For example, the first heater 134 is controlled by the first heater driver 130 to maintain the temperature of the reaction region on the right side of the paper in the thermal circulation region 12e of the flow channel 12 at a fixed 94°C. Furthermore, the second heater driver 132 controls the second heater 135 to maintain the temperature of the reaction region on the left side of the paper at a fixed 60°C. Alternatively, the temperature of each reaction region can be measured by a temperature sensor (not shown) such as a thermocouple, and each driver controls the output to each heater based on its electrical signal. Thus, the first heater 134, the second heater 135, the first heater driver 130, the second heater driver 132, and the temperature sensor constitute a temperature control unit for regulating the temperature of the thermal cycling region 12e, but it may also include other elements for improving temperature controllability. Hereinafter, the reaction region in the flow channel 12 with an atmosphere temperature of 94°C will be referred to as the "high-temperature region 111," and the reaction region in the flow channel 12 with an atmosphere temperature of 60°C will be referred to as the "medium-temperature region 112." Furthermore, this embodiment details a PCR apparatus having a PCR reaction vessel and a temperature control unit, wherein the PCR reaction vessel has a thermal cycling region capable of setting two standard temperature regions as two reaction regions, but it may also be a PCR apparatus having a PCR reaction vessel and a temperature control unit, wherein the PCR reaction vessel has a thermal cycling region capable of setting three or more standard temperature regions. At this time (not shown), as an example, a PCR apparatus having a PCR reaction vessel and a temperature control unit may be such that the PCR reaction vessel has reaction regions arranged from the left side of the paper in the order of low-temperature region, medium-temperature region, and high-temperature region. In this case, for example, the temperature is controlled to maintain the low-temperature section at 50-70°C, the medium-temperature section at 72°C, and the high-temperature section at 94°C.

[0080] As described above, the pump system 110 is configured to reciprocate the sample 70 within the thermal cycling region 12e of the flow channel 12. By having the first pump 103 and the second pump 104 operate alternately under certain conditions via the first driver 105 and the second driver 106 through the control unit 107, the sample 70 can reciprocate between the high-temperature section 111 and the intermediate-temperature section 112 of the flow channel 12, thereby enabling thermal cycling of the sample 70 under certain conditions. In the PCR apparatus 100 according to this first embodiment, the first pump 103 and the second pump 104 are air pumps or booster pumps of the type such that when they are both stopped, the air pressure on the primary side and the secondary side becomes equal instantaneously; furthermore, when they are both stopped, the air pressure on the primary side and the secondary side becomes equal. If this type of pump is not used, that is, if a pump that maintains the pressure it was about to stop even when stopped is used, the sample may continue to move slightly even after the pump stops, and therefore will not stop in the predetermined reaction area, resulting in inadequate control of the sample temperature. Furthermore, in the PCR apparatus 100 according to this first embodiment, when stopped (open), the external air is connected to the flow channel pressure of the PCR reaction vessel, becoming equal to atmospheric pressure. However, since a filter is provided between the air inlet and the flow channel, contamination into the flow channel can be prevented.

[0081] Sample 70 can be subjected to PCR through the thermal cycling described above. Fluorescence from sample 70 within the flow channel can be detected, and its value can be used as an indicator of PCR progress or reaction termination. The optical detector 122 and driver 121 for fluorescence detection can be a fiber optic fluorescence detector FLE-510 manufactured by Nippon Sheet Glass Co., Ltd., which can perform rapid measurements with a very compact optical system and detect fluorescence regardless of ambient light or darkness. This fiber optic fluorescence detector can also be easily configured within the narrow space between the two reaction zones in the thermal cycling region. The wavelength characteristics of its excitation light / fluorescence can be pre-tuned to suit the fluorescence characteristics of sample 70, providing the most suitable optical detection system for samples with various characteristics. Furthermore, multiple optical detectors 122 and drivers 121 for fluorescence detection can be provided within the thermal cycling region 12e. For example, they can be configured to detect fluorescence from sample 70 located in the flow channels of the high-temperature section 111 and the intermediate-temperature section 112. It not only has the function of obtaining materials for judging the progress and end of PCR, but also can be used as a position sensor. The position sensor can reliably detect whether the sample 70 is located in the high temperature section 111 or the medium temperature section 112.

[0082] In the PCR apparatus 100 configured as described above, the control unit 107 of the pump system 110, the driver 121 of the fluorescence detection optical detector 122, the first heater driver 130, and the second heater driver 132 are all controlled by the CPU 141 to operate optimally. Furthermore, as mentioned above, when the reaction zone has three standard temperatures, in addition to the above, the third heater driver (not shown) is also controlled by the CPU.

[0083] Figure 10 This diagram illustrates the connection between the nozzle of the pump system and the air vent of the PCR reaction vessel. Figure 11 yes Figure 10 The diagram shows a cross-sectional view of the PCR reaction vessel 10. As described above, the first nozzle 101 is connected to the first air vent 24, and the second nozzle 102 is connected to the second air vent 26.

[0084] like Figure 11 As shown, a hollow needle 150 is provided at the front end of the first nozzle 101. The first nozzle 101 is connected to the first air vent 24 by perforating the first sealing film 18 through the needle 150. The connection between the second nozzle 102 and the second air vent 26 is the same.

[0085] In the needle 150, a sealing element 151 made of soft resin that adheres to the surface of the sealing film is provided to ensure the airtightness around the connection part. Since the pump system 110 is not yet working and is open to the atmosphere when the PCR reaction container 10 is first set in the PCR device 100, the pressure in the flow channel is equal to the atmospheric pressure.

[0086] Figure 12 This indicates the situation where the pump system 110 is activated to move the sample 70. Either the first pump 103 or the second pump 104 is activated to move the sample 70 toward the high-temperature section 111 or the medium-temperature section 112 of the thermal cycling region 12e. Figure 12 In this process, the second pump 104 connected to the second nozzle 102 is activated, while the first pump 103 connected to the first nozzle 101 is deactivated. That is, the first air vent 24 connected to the first nozzle 101, extending from the first pump 103, is opened to atmospheric pressure. The second pump 104 is activated so that when air is introduced from the second nozzle 102 into the second air vent 26, the sample 70 moves, i.e., it moves from the intermediate temperature section 112 to the high temperature section 111. This state is referred to as the initial state.

[0087] More specifically, the fluorescence detection optical detector 122 is used simultaneously with the start of operation of the second pump 104, or just before its start, to begin monitoring the fluorescence emitted from the sample in the flow channel. When there is nothing at the measurement point of the fluorescence detection optical detector 122, the detected fluorescence is zero or at the background level; however, when the sample 70 is present at the measurement point, fluorescence is detected. Therefore, fluorescence monitoring begins before the second pump 104 starts operating, allowing the sample 70 to be detected by observing the fluorescence rise from the background level and then fall back to the background level. At this point, the operation of the second pump 104 is stopped, thus completing the initial setting. Furthermore, if the fluorescence detection optical detector is still located in the high-temperature section 111, the sample 70 can be more reliably stopped in the high-temperature section 111.

[0088] Note that even when the second pump 104 is operating, the sample 70 located in the bifurcation channel 131 will almost remain there. This is because the sample inlet 133 is sealed by the third sealing film 22. The sample 70 located in this bifurcation channel 131 is not used for PCR.

[0089] After setting to the initial state, sample 70 was thermally cycled for PCR. Fluorescence measurement based on optical detector 122 was then performed.

[0090] (A) First, sample 70 is placed in high-temperature section 111 (at an atmosphere of approximately 94°C) for 1–30 seconds (Deneturization). Through this process, double-stranded DNA is denatured into single-stranded DNA.

[0091] (B) Next, the first pump 103 connected to the first nozzle 101 is activated to move the sample 70 to the intermediate temperature section 112 (an atmosphere of approximately 60°C). Specifically, the sample 70 is propelled from the high temperature section 111 towards the intermediate temperature section 112 by the action of the first pump 103. Since the fluorescence measurement performed by the fluorescence detection optical detector 122 is still ongoing, the operation of the first pump 103 is stopped when the fluorescence intensity rises from the background level and then decreases after the sample 70 passes the measurement point of the fluorescence detection optical detector 122 (or after a period of time has elapsed after the fluorescence intensity decreases). Furthermore, if the fluorescence detection optical detector 122 is present in the intermediate temperature section 112, the sample 70 can be stopped in the intermediate temperature section 112 more reliably.

[0092] (C) The sample 70 is held in the intermediate temperature section 112 for 3 to 60 seconds (Annealing + Elongation). Through this process, the primers contained in the sample 70 bind and become extended DNA.

[0093] (D) Next, the second pump 104 connected to the second nozzle 102 is activated to move the sample 70 from the intermediate temperature section 112 to the high temperature section 111. As described above, the pump operation is stopped based on the change in fluorescence intensity measured by the fluorescence detection optical detector 122. After the sample 70 is moved to the high temperature section 111, it is held for 1 to 30 seconds to allow it to undergo thermal denaturation.

[0094] (E) Repeat steps (B) to (D) above for a predetermined number of rounds to thermally cycle sample 70, so that the DNA contained in sample 70 undergoes multiple rounds of thermal denaturation-annealing-extension processes to achieve DNA amplification. The number of rounds is appropriately determined based on the combination of the target DNA and primers, enzymes, etc.

[0095] After the predetermined number of thermal cycles, pumps 103 and 104 are stopped to terminate the PCR. During the predetermined number of thermal cycles, the fluorescence detection optical detector 122 continues to measure fluorescence; the fluorescence detected from sample 70 increases with the amplification of the DNA contained in sample 70. This allows for accurate determination of the concentration of sample 70.

[0096] According to the PCR reaction container 10 of the first embodiment, by providing a first filter 28 between the first air vent 24 and the flow channel 12, and a second filter 30 between the second air vent 26 and the flow channel 12, contamination within the flow channel 12 can be prevented. While implementing contamination prevention measures on the pump system 110 side often increases costs, the PCR reaction container 10 of this first embodiment saves costs because contamination can be prevented only on the PCR reaction container 10 side. Furthermore, if the PCR reaction container is used as a disposable component, since the filters are always new, further cost-effective contamination prevention can be achieved. Moreover, regarding the disposal of the PCR reaction container, since the sample is in a substantially sealed state within the PCR reaction container, this is significant from both safety and environmental protection perspectives.

[0097] In the PCR apparatus 100 according to this first embodiment, by alternating operation of the first pump 103 and the second pump 104, which make the pressure on the primary side and the secondary side equal when stopped, the sample can be moved back and forth in the flow channel 12 of the PCR reaction vessel 10. At this time, the sample in the liquid delivery (applying pressure to the sample in the flow channel) is not subjected to excessive pressure, and no decompression is performed in the flow channel, so it is possible to prevent the evaporation or boiling (foaming) of the liquid containing the sample due to the action of the high temperature section 111.

[0098] Furthermore, in the PCR apparatus 100 according to this first embodiment, fluorescence from the sample is continuously monitored during the PCR process in the thermal cycling zone (real-time PCR). Therefore, the timing for the termination of PCR can be determined based on the measured fluorescence intensity. Moreover, by monitoring changes in fluorescence using the fluorescence detection optical detector 122, the passage of the sample can be detected, and the alternating operation of the first pump 103 and the second pump 104 can be controlled based on the changes in fluorescence intensity accompanying this passage. Thus, the sample supplied for PCR can be accurately positioned in the high-temperature section 111 or the intermediate-temperature section 112 of the thermal cycling zone.

[0099] Furthermore, when using PCR reaction containers and PCR apparatuses with reaction regions controlled to the aforementioned three standard temperatures—high-temperature, medium-temperature, and low-temperature—the processes of thermal denaturation in the high-temperature region, annealing in the medium-temperature region, and extension in the low-temperature region can be easily extended and improved by those skilled in the art based on the above detailed description. Moreover, those skilled in the art can appropriately select whether to set the reaction regions to two or three standards depending on the characteristics of the sample.

[0100] [Second Implementation]

[0101] Figure 13 (a) and (b) are diagrams illustrating the PCR reaction vessel 210 according to the second embodiment of the present invention. Figure 13 (a) is a top view of PCR reaction vessel 210. Figure 13 (b) is a front view of PCR reaction vessel 210. Figure 14 yes Figure 13 (a) shows a cross-sectional view of the PCR reaction vessel 210. Figure 15 yes Figure 13 (a) shows a BB cross-section of the PCR reaction vessel 210. Figure 16 This is a top view of the substrate 214 of the PCR reaction vessel 210. Figure 17 This is a schematic diagram illustrating the structure of the PCR reaction container 210. The PCR reaction container 210 in the second embodiment has two bifurcation points (first bifurcation point 212c and second bifurcation point 212d) and two bifurcation channels extending from these points, as well as sample inlets (first bifurcation channel 231 and first sample inlet 233, second bifurcation channel 232 and second sample inlet 234), and differs from the first embodiment in that it has a buffer channel region 212f between the first bifurcation point 212c and the second bifurcation point 212d.

[0102] The PCR reaction vessel 210 is composed of the following components: a resin substrate 214 with grooved flow channels 212 formed on the bottom surface 214a; a flow channel sealing film 216, which is attached to the bottom surface 214a of the substrate 214 and is used to seal the flow channels 212; and three sealing films (a first sealing film 218, a second sealing film 220, and a third sealing film 222) which are attached to the top surface 214b of the substrate 214.

[0103] The substrate 214 is preferably formed of a material with good thermal conductivity, stable performance relative to temperature changes, and low permeability to the sample solution used. Furthermore, the substrate 214 is preferably formed of a material with high plasticity, good transparency and insulation, and weak autofluorescence. As such a material, inorganic materials such as glass and silicon are preferred, as well as resins such as acrylic resin, polyester, and silicone, with cycloolefins being particularly preferred. As an example of the dimensions of the substrate 214, the long side is 70 mm, the short side is 42 mm, and the thickness is 3 mm. As an example of the dimensions of the flow channel 212 formed on the bottom surface 214a of the substrate 214, the width is 0.5 mm and the depth is 0.5 mm.

[0104] As described above, a groove-shaped flow channel 212 is formed on the bottom surface 214a of the substrate 214, and the flow channel 212 is sealed by a flow channel sealing film 216 (see reference). Figure 14 A first air vent 224 is formed at one end 212a of the flow channel 212 in the substrate 214. A second air vent 226 is formed at the other end 212b of the flow channel 212 in the substrate 214. The pair of first air vents 224 and second air vents 226 are formed to protrude from the top surface 214b of the substrate 214. Such a substrate can be manufactured by injection molding or machining using an NC machining machine.

[0105] A first filter 228 is provided between the first air vent 224 in the substrate 214 and one end 212a of the flow channel 212 (see reference). Figure 14 A second filter 230 is provided between the second air vent 226 in the substrate 214 and the other end 212b of the flow channel 212. The pair of first filters 228 and second filters 230 provided at both ends of the flow channel 212 have good low-impurity characteristics and allow only air to pass through, thereby preventing contamination and ensuring that the quality of the DNA amplified by PCR does not deteriorate. As the filter material, polyethylene or PTFE is preferred, and it may also be porous or hydrophobic. The dimensions of the first filter 228 and the second filter 230 are formed so that they can be precisely accommodated in the filter placement space formed on the substrate 214.

[0106] In substrate 214, a first branched flow channel 231 is formed at the first branch point 212c between the first filter 228 and the second filter 230, branching off from the flow channel 212. At the end 231a of the first branched flow channel 231 in substrate 214, a first sample inlet 233 is formed (see reference). Figure 15 In substrate 214, a second branched flow channel 232 branching out from flow channel 212 is formed at a second branched point 212d located between the first branched point 212c and the second filter 230. A second sample inlet 234 is provided at the end 232a of the second branched flow channel 232 in substrate 214. The first sample inlet 233 and the second sample inlet 234 are formed to expose the top surface 214b of substrate 214.

[0107] The portion of the flow channel 212 between the first filter 228 and the first bifurcation point 212c forms a thermal cycling region 212e for applying thermal cycling to the sample. This thermal cycling region 212e is pre-defined to include a high-temperature region and a medium-temperature region. The thermal cycling region 212e of the flow channel 212 includes a serpentine flow path. This is to efficiently transfer the heat provided by the PCR device during the PCR process to the sample, and to ensure that the volume of sample available for PCR is sufficient. The thermal cycling region 212e has a pair of reaction regions, each including a serpentine flow path, and a connecting region connecting the pair of reaction regions.

[0108] The portion of flow channel 212 between the first bifurcation point 212c and the second bifurcation point 212d forms a buffer flow channel region 212f. The buffer flow channel region 212f of flow channel 212 includes a serpentine flow path. The volume of the buffer flow channel region 212f of flow channel 212 is set to a predetermined volume corresponding to the amount of sample to be subjected to PCR treatment. The function of the buffer flow channel region will be described below.

[0109] In the PCR reaction vessel 210 according to this second embodiment, most of the flow channel 212 is formed as a groove exposed on the bottom surface 214a of the substrate 214. This is to facilitate easy molding using injection molding or the like. To utilize this groove as a flow channel, a flow channel sealing film 216 is adhered to the bottom surface 214a of the substrate 214. The flow channel sealing film 216 may have adhesive properties on one main surface, or it may have a functional layer formed on one main surface that can exert adhesiveness or bonding by pressing, thereby enabling it to be easily adhered to and integrated with the bottom surface 214a of the substrate 214. The flow channel sealing film 216 is preferably formed of a material with weak autofluorescence, including the adhesive. In this regard, a transparent film made of resins such as cyclic olefin polymers, polyesters, polypropylene, polyethylene, or acrylic resins is more suitable, but not limited to this. Alternatively, the flow channel sealing film 216 may also be formed of sheet-like glass or resin. At this point, due to its rigidity, it can effectively prevent the PCR reaction container 210 from warping or deforming.

[0110] Furthermore, in the PCR reaction vessel 210 according to this second embodiment, the first air vent 224, the second air vent 226, the first sample inlet 233, the second sample inlet 234, the first filter 228, and the second filter 230 are all exposed on the top surface 214b of the substrate 214. Therefore, to seal the first air vent 224 and the first filter 228, a first sealing film 218 is attached to the top surface 214b of the substrate 214. Furthermore, to seal the second air vent 226 and the second filter 230, a second sealing film 220 is attached to the top surface 214b of the substrate 214. Furthermore, to seal the first sample inlet 233 and the second sample inlet 234, a third sealing film 222 is attached to the top surface 214b of the substrate 14.

[0111] The first sealing film 218 is a sealing film large enough to simultaneously seal the first air vent 224 and the first filter 228, and the second sealing film 220 is a sealing film large enough to simultaneously seal the second air vent 226 and the second filter 230. The connection of the pressure pump (described below) to the first air vent 224 and the second air vent 226 is achieved by a hollow needle (a sharp-tipped injection needle) provided at the front end of the pump penetrating the first air vent 224 and the second air vent 226. Therefore, the first sealing film 218 and the second sealing film 220 are preferably films made of a material and thickness that are easily perforated by a needle. This second embodiment describes a sealing film large enough to simultaneously seal the corresponding air vent and filter, but it is also possible to seal them separately. Alternatively, a sealing film capable of sealing the first air vent 224, the first filter 228, the second air vent 226, and the second filter 230 together (on a single sheet) is also possible.

[0112] The third sealing film 222 is a sealing film that can simultaneously seal both the first sample inlet 233 and the second sample inlet 234. The sample is introduced into the flow channel 212 through the first sample inlet 233 and the second sample inlet 234 by temporarily peeling the third sealing film 222 off the substrate 214. After a predetermined amount of sample is introduced, the third sealing film 222 is then adhered back to the top surface 214b of the substrate 214. Therefore, the third sealing film 222 is preferably a film with adhesiveness capable of withstanding several rounds of adhesion and peeling. Furthermore, the third sealing film 222 can also be a new film adhered after the sample is introduced; in this case, the importance of adhesion and peeling characteristics can be reduced. Furthermore, while this second embodiment describes a sealing film that can simultaneously seal both the first sample inlet 233 and the second sample inlet 234, it is also possible to seal them separately.

[0113] The first sealing film 218, the second sealing film 220, and the third sealing film 222, like the flow channel sealing film 216, may have an adhesive layer formed on one of their main surfaces, or a functional layer capable of exerting adhesion or bonding through pressing. Preferably, the first sealing film 218, the second sealing film 220, and the third sealing film 222, including the adhesive, are all formed of a material with weak self-fluorescence. In this regard, transparent films made of resins such as cycloolefins (COP), polyesters, polypropylene, polyethylene, or acrylic resins are suitable, but not limited to these. Furthermore, it is preferable that, as described above, even after repeated application and peeling, the adhesive properties do not deteriorate to the point of affecting its use. However, in cases where a new film is applied after peeling and introducing a sample, the importance of these adhesion and peeling properties can be reduced.

[0114] Next, the method of using the PCR reaction vessel 210 constructed as described above will be explained. First, prepare the sample to be amplified by thermal cycling. Examples of samples include products obtained by adding various primers, thermostable enzymes, and four deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP) as PCR reagents to a mixture containing two or more DNAs. Next, peel the third sealing film 222 off the substrate 214 to open the first sample inlet 233 and the second sample inlet 234.

[0115] Next, use a pipette or syringe to introduce a sample into either the first sample inlet 233 or the second sample inlet 234. Figure 18 This schematically illustrates the introduction of sample 270 into PCR reaction vessel 210. Furthermore, Figure 18 In the diagram, to highlight the position of sample 270, a solid line thicker than flow channel 212 is used to represent sample 270. Note that this does not indicate that sample 270 is overflowing from the flow channel. Figure 18 As shown, the sample 270, introduced into either the first sample inlet 233 or the second sample inlet 234, fills the flow channel by being forced in by a suction tube or syringe, or by capillary action. The sample 270 is filled in the buffer flow channel region 212f between the first bifurcation point 212c and the second bifurcation point 212d in the flow channel 212. However, the sample 270 does not cross the first bifurcation point 212c and the second bifurcation point 212d located at both ends of the buffer flow channel region and intrude into the thermal circulation region 212e and the area of ​​the second air vent 26 of the flow channel 212. This is because both ends of the flow channel (i.e., the first air vent 224 and the second air vent 226) are sealed at this time, and there is no air outlet.

[0116] Next, as Figure 19 As shown, the third sealing film 222 is then pasted back onto the substrate 214 to seal the first sample inlet 233 and the second sample inlet 234. Alternatively, a new third sealing film 222 can be pasted as described above. This completes the process of introducing sample 270 into the PCR reaction vessel 210.

[0117] Figure 20 This is a diagram illustrating a PCR apparatus 300, in which a PCR reaction vessel 210 is used. Figure 21 This is a state diagram used to illustrate the placement of the PCR reaction vessel 210 in the predetermined position of the PCR apparatus 300.

[0118] The PCR apparatus 300 includes a fluorescence detection optical detector 2122, a first heater 2134, and a second heater 2135. For example... Figure 21 As shown, regarding the PCR reaction vessel 210, a pair of reaction regions of the thermal cycling region 212e of its flow channel 212 are disposed on the first heater 2134 and the second heater 2135, and a fluorescence detection optical detector 2122 is disposed in the connection region between the pair of reaction regions in the PCR apparatus 300. The PCR apparatus 300 can refer to the PCR apparatus applicable to the PCR reaction vessel according to the first embodiment.

[0119] The PCR apparatus 300 also includes a pump system 2110 for reciprocating motion of the sample 270 within the thermal cycling zone 212e. This pump system 2110 includes a first nozzle 2101, a second nozzle 2102, a first pump 2103, a second pump 2104, a first actuator 2105, a second actuator 2106, and a control unit 2107. The first nozzle 2101 of the pump system 2110 is connected to the first air vent 224 of the PCR reaction vessel 210, and the second nozzle 2102 of the PCR reaction vessel 210 is connected to the second air vent 226 of the PCR reaction vessel 210. The specific connection method between the nozzles and the air vents will be described below. The pump system 2110 moves the sample within the thermal cycling zone 212e by controlling the pressure within the flow channel 212 through the first air vent 224 and the second air vent 226.

[0120] In the PCR apparatus 300 according to this second embodiment, the first heater 2134 and the second heater 2135 are set to different temperatures. Each heater is a component that supplies heat to a pair of reaction regions in the thermal circulation region 212e, and may be a resistance heater or a Peltier element, or similar means or structure. For example, the first heater driver 2130 controls the first heater 2134 so that the temperature of the reaction region on the right side of the paper in the thermal circulation region 212e of the flow channel 212 is maintained at a fixed 94°C. Furthermore, the second heater driver 2132 controls the second heater 2135 so that the temperature of the reaction region on the left side of the paper is maintained at a fixed 60°C. This can be achieved by measuring the temperature of each reaction region using a temperature sensor (not shown) such as a thermocouple, and by each driver controlling the output to each heater based on its electrical signal. Thus, the first heater 2134, the second heater 2135, the first heater driver 2130, the second heater driver 2132, and the temperature sensor constitute a temperature regulating unit for adjusting the temperature of the thermal circulation region 212e, but it may also include other elements for improving temperature controllability. This temperature regulating unit can divide the thermal circulation region 212e of the flow channel 212 into two regions with different atmosphere temperatures. Each heater may contain a temperature sensor (not shown), such as a thermocouple, for measuring the temperature at the corresponding location, or other structures for improving temperature controllability. Hereinafter, the reaction region in the flow channel 212 with an atmosphere temperature of 94°C will be referred to as the "high-temperature region 2111," and the reaction region in the flow channel 212 with an atmosphere temperature of 60°C will be referred to as the "low-temperature region 2112." Furthermore, this embodiment details a PCR apparatus having a PCR reaction vessel and a temperature control unit. The PCR reaction vessel has a thermal cycling region capable of setting two standard temperature zones as two reaction zones. However, the PCR apparatus may also have a PCR reaction vessel and a temperature control unit where the PCR reaction vessel has a thermal cycling region capable of setting three or more standard temperature zones. As an example (not shown), a PCR apparatus with a PCR reaction vessel and a temperature control unit could be described as follows: the PCR reaction vessel has reaction zones arranged from the left side of the paper in the order of low temperature zone, intermediate temperature zone, and high temperature zone. In such a case, for example, the low temperature zone is controlled to be maintained at 50–70°C, the intermediate temperature zone at 72°C, and the high temperature zone at 94°C.

[0121] As described above, the pump system 21100 is configured to reciprocate the sample 270 within the thermal cycling region 212e of the flow channel 212. The control unit 2107, via the first driver 2105 and the second driver 2106, causes the first pump 2103 and the second pump 2104 to operate alternately under certain conditions. This allows the sample 270 to reciprocate between the high-temperature section 2111 and the intermediate-temperature section 2112 of the flow channel 212, thereby subjecting the sample 270 to thermal cycling under certain conditions. In the PCR apparatus 300 according to this second embodiment, the first pump 2103 and the second pump 2104 are air pumps or booster pumps of the type such that when they are both stopped, the air pressure on the primary side and the secondary side becomes equal instantaneously; furthermore, when they are both stopped, the air pressure on the primary side and the secondary side becomes equal. If this type of pump is not used—that is, if a pump that maintains the pressure it was about to stop even after stopping is used—the sample may continue to move slightly even after the pump stops, and therefore will not stop in the intended reaction area, resulting in inadequate temperature control of the sample. Furthermore, during shutdown (opening), the external air pressure is connected to the flow channel of the PCR reaction vessel, becoming equal to atmospheric pressure. However, because there is a filter between the air inlet and the flow channel, contamination into the flow channel is prevented.

[0122] Sample 270 can undergo PCR through the aforementioned thermal cycling, and the fluorescence from sample 270 within the flow channel can be detected, with its value used as an indicator of PCR progress or reaction termination. The optical detector 2122 and driver 2121 for fluorescence detection can be the fiber optic fluorescence detector FLE-510 manufactured by Nippon Sheet Glass Co., Ltd., which allows for rapid measurement with a very compact optical system and can detect fluorescence regardless of ambient light or darkness. This fiber optic fluorescence detector can also be easily configured within a narrow space between two temperature zones within the thermal cycling region. The wavelength characteristics of its excitation light / fluorescence can be pre-adjusted to suit the fluorescence characteristics of sample 270, providing the most suitable optical and detection system for samples with different characteristics. Furthermore, multiple optical detectors 2122 and drivers 2121 for fluorescence detection can be provided within the thermal cycling region 212e. For example, they can be configured to detect fluorescence from sample 270 located in the flow channel of the high-temperature section 2111 or the intermediate-temperature section 2112. It not only has the function of obtaining materials for judging the progress and end of PCR, but also can be used as a position sensor. The position sensor can reliably detect whether the sample 270 is located in the high temperature section 2111 or the medium temperature section 2112.

[0123] In the PCR apparatus 300 configured as described above, the control unit 2107 of the pump system 2110, the driver 2121 of the fluorescence detection optical detector 2122, the first heater driver 2130, and the second heater driver 2132 are controlled by the CPU 2141 to operate optimally. Furthermore, as mentioned above, when the reaction zone has three standard temperatures, in addition to the above, the third heater driver (not shown) is also controlled by the CPU.

[0124] Figure 22 This diagram illustrates the connection between the nozzle of the pump system and the air vent of the PCR reaction vessel. Figure 23 Is with Figure 23 The diagram shows a cross-sectional view of the PCR reaction vessel 210. As described above, the first nozzle 2101 is connected to the first air vent 224, and the second nozzle 2102 is connected to the second air vent 226.

[0125] like Figure 23 As shown, a needle 2150 is provided at the front end of the first nozzle 2101. The first nozzle 2101 is connected to the first air vent 224 by the needle 2150 perforating the first sealing film 218. The connection between the second nozzle 2102 and the second air vent 226 is the same.

[0126] To ensure airtightness around the connection point, a sealing element 2151 made of soft resin is provided on the needle 2150, which adheres to the surface of the sealing film. Since the pump system 2110 is not yet working and is open to the atmosphere when the PCR reaction vessel 210 is first placed in the PCR device 300, the pressure in the flow channel is equal to the atmospheric pressure.

[0127] Figure 24 This indicates the situation where the pump system 2110 is operated to move the sample 270. Either the first pump 2103 or the second pump 2104 is operated to move the sample 270 from the buffer flow channel region 212f of the flow channel 212 to the high-temperature section 2111 or the medium-temperature section 2112 of the thermal cycling region 212e. Figure 24 In this process, the second pump 2104 connected to the second nozzle 2102 is activated, but the first pump 2103 connected to the first nozzle 2101 is stopped. That is, the first air vent 224 connected to the first nozzle 2101, which extends from the first pump 2103, is opened to atmospheric pressure. When the second pump 2104 is activated so that air is introduced from the second nozzle 2102 into the second air vent 226, the sample 270 moves from the buffer flow channel region 212f of the flow channel 212, passes through the intermediate temperature section 2112, and moves to the high temperature section 2111. This state is called the initial state.

[0128] More specifically, the fluorescence detection optical detector 2122 is used simultaneously with the start of operation of the second pump 2104, or just before the start of operation of the second pump 2104, to monitor the fluorescence emitted from the flow channel. If there is nothing at the measurement point of the fluorescence detection optical detector 2122, the detected fluorescence is zero or at the background level; however, if the measurement point contains sample 270, fluorescence will be detected. Therefore, by monitoring fluorescence from the start of operation of the second pump 2104, and by observing the fluorescence value rise from the background level and then fall back to the background level, it can be determined that sample 270 has moved to the high-temperature section 2111. At this point, the operation of the second pump 2104 is stopped, thus completing the initial state setup. Furthermore, if the fluorescence detection optical detector 2122 is also located in the high-temperature section 2111, the sample 270 can be more reliably stopped in the high-temperature section 2111.

[0129] Here, please note that even when the second pump 2104 is operating, the sample 270 located in the first branch channel 231 and the second branch channel 232 will remain there. This is because the first sample inlet 233 and the second sample inlet 234 are sealed by the third sealing film 222. The sample 270 located in the first branch channel 231 and the second branch channel 232 is not used for PCR. Therefore, even if there is a deviation in the amount of sample initially introduced into the PCR reaction vessel 210, the desired amount of sample can always be delivered into the thermal cycling region 212e of the channel 212 by setting the volume of the buffer channel region 212f formed in the PCR reaction vessel 210 to a predetermined volume corresponding to the amount of sample to be PCR treated. This allows the fluorescence intensity that affects the determination of the progress or end of PCR to be approximately fixed. That is, the buffer flow channel region 212f of the flow channel 212 has a dispensing function that can extract a certain amount of sample.

[0130] After setting to the initial state, thermal cycling was applied to sample 270 to advance PCR. Fluorescence measurement based on optical detector 2122 for fluorescence detection was then performed.

[0131] (A) First, the sample 270 is placed in the high-temperature section 2111 (at an atmosphere of approximately 94°C) for 1 to 30 seconds (Deneturation). Through this process, the double-stranded DNA is denatured into single strands.

[0132] (B) Next, the first pump 2103 connected to the first nozzle 2101 is activated to move the sample 270 to the intermediate temperature section 2112 (an atmosphere of approximately 60°C). Specifically, the sample 270 is propelled from the high temperature section 2111 to the intermediate temperature section 2112 by the action of the first pump 2103. Since the fluorescence measurement performed by the fluorescence detection optical detector 2122 is still ongoing, the operation of the first pump 2103 is stopped when the fluorescence intensity rises from the background level and then decreases after the sample 270 passes the measurement point of the fluorescence detection optical detector 2122 (or after a certain period of time has elapsed after the fluorescence intensity decreases). Furthermore, if the fluorescence detection optical detector 2122 is located in the intermediate temperature section 2112, the sample 270 can be stopped in the intermediate temperature section 2112 more reliably.

[0133] (C) In the intermediate temperature section 2112, the sample 270 is held for 3 to 60 seconds (Annealing + Elongation). Through this process, the primers previously contained in the sample 270 bind, thus becoming extended DNA.

[0134] (D) Next, the second pump 2104 connected to the second nozzle 2102 is activated to move the sample 270 from the intermediate temperature section 2112 to the high temperature section 2111. As described above, the pump operation is stopped based on the change in fluorescence intensity measured by the fluorescence detection optical detector 2122. After the sample 270 is moved to the high temperature section 2111, it is held for 1 to 30 seconds to allow it to undergo thermal denaturation.

[0135] (E) Repeat steps (B) to (D) above for a predetermined number of rounds to thermally cycle sample 270, causing the DNA contained in sample 270 to undergo multiple rounds of thermal denaturation-annealing-extension processes to achieve DNA amplification. The number of rounds is appropriately determined based on the combination of the target DNA and primers, enzymes, etc.

[0136] After the predetermined number of thermal cycles, pumps 1 (2103) and 2 (2104) are stopped to terminate the PCR. During the predetermined number of thermal cycles, the fluorescence detection optical detector 122 continues to measure fluorescence; the fluorescence detected from sample 270 increases with the amplification of the DNA contained in sample 270. This allows for accurate determination of the concentration of sample 270.

[0137] According to the PCR reaction vessel 210 of this second embodiment, by providing a first filter 228 between the first air vent 224 and the flow channel 212, and a second filter 230 between the second air vent 226 and the flow channel 212, contamination towards the flow channel 212 can be prevented. While implementing contamination prevention measures on the pump system 2110 side often increases costs, the PCR reaction vessel 210 of this second embodiment saves costs because contamination can be prevented only on the PCR reaction vessel 210 side. Furthermore, if the PCR reaction vessel is used as a disposable component, since the filters are always new, further low-cost contamination prevention can be achieved. Moreover, regarding the disposal of the PCR reaction vessel, since the sample is in a substantially sealed state within the PCR reaction vessel, this is significant from both safety and environmental perspectives.

[0138] Furthermore, according to the PCR reaction vessel 210 of this second embodiment, by providing a buffer flow channel region in the flow channel 212 for dispensing samples for PCR, it is always possible to deliver only the necessary amount of sample into the thermal cycling region of the flow channel 212.

[0139] In the PCR apparatus 300 according to this second embodiment, by having the first pump 2103 and the second pump 2104 operate alternately so that the pressure on the primary side and the secondary side becomes equal when stopped, the sample can be moved back and forth within the flow channel 212 of the PCR reaction vessel 210. At this time, since the sample in the liquid delivery (applying pressure to the sample in the flow channel) is not subjected to excessive pressure, and no decompression is performed in the flow channel, evaporation or boiling (foaming) of the liquid containing the sample due to the action of the high-temperature section 2111 can be prevented.

[0140] Furthermore, in the PCR apparatus 300 according to this second embodiment, fluorescence from the sample is continuously monitored during the PCR process in the thermal cycling zone (real-time PCR). Therefore, the timing for the termination of PCR can be determined based on the measured fluorescence intensity. Moreover, the sample's passage can be known by monitoring changes in fluorescence using the fluorescence detection optical detector 2122, and the alternating operation of the first pump 2103 and the second pump 2104 can be controlled based on changes in fluorescence intensity accompanying this passage. Thus, the sample supplied for PCR can be accurately positioned in the high-temperature section 2111 or the intermediate-temperature section 2112 of the thermal cycling zone.

[0141] Furthermore, when using a PCR reaction vessel and PCR apparatus with reaction zones controlled at three standard temperatures—a high-temperature zone, a medium-temperature zone, and a low-temperature zone—it is possible to enable the high-temperature zone to undergo thermal denaturation, the medium-temperature zone to undergo annealing, and the low-temperature zone to undergo extension processes. These controls are matters that can be easily extended and improved by those skilled in the art based on the above detailed description.

[0142] Furthermore, those skilled in the art can appropriately choose whether to set the reaction region to two or three standards based on the characteristics of the sample.

[0143] The present invention has now been described based on embodiments. These embodiments are illustrative, and those skilled in the art will understand that various modifications can be made to these constituent elements or combinations of processes, and such modifications are also included within the scope of the present invention.

[0144] In the above embodiment, a pair of pumps are arranged at both ends of the flow channel so that the pressure on the primary and secondary sides becomes equal when the flow channel stops. However, it is also possible to provide a pump capable of pressurization and suction at only one end of the flow channel, with the other end open to atmospheric pressure. That is, the pressure within the flow channel is controlled via a first air vent or a second air vent to allow the sample to move within the thermal cycling region. In this case, the process of switching the operation of one pair of pumps at fixed times is not required, thus simplifying pump control.

[0145] Furthermore, in the above embodiment, the measurement point of the fluorescence detection optical detector is arranged between the high-temperature section and the intermediate-temperature section. However, the measurement point of the fluorescence detection optical detector can also be arranged separately in the high-temperature section and the intermediate-temperature section. In this case, the positioning accuracy of the sample can be improved.

[0146] [Label Explanation]

[0147] 10, 210 PCR reaction vessel; 12, 212 flow channels; 14, 214 substrate; 16, 216 flow channel sealing film; 18, 218 first sealing film; 20, 220 second sealing film; 22, 222 third sealing film; 24, 224 first air vent; 26, 226 second air vent; 28, 228 first filter; 30, 230 second filter; 70, 270 sample; 100, 300 PCR apparatus, 101, 2101 Nozzle 1, 102, 2102 Nozzle 2, 103, 2103 Pump 1, 104, 2104 Pump 2, 105, 2105 Driver 1, 106, 2106 Driver 2, 107, 2107 Control unit, 110, 2110 Pump system, 111, 2111 High-temperature section, 112, 2112 Intermediate-temperature section, 121, 2121 Driver, 122, 2122 Optical detector for fluorescence detection, 130, 2130 Driver 1 Heater, 131 Bifurcation channel, 132, 2132 Driver 2 Heater, 133 Sample inlet, 134, 2134 Heater 1, 135, 2135 Heater 2, 141, 2141 CPU, 231 First branch flow channel, 232 Second branch flow channel, 233 First sample inlet, 234 Second sample inlet.

[0148] [Industrial Availability]

[0149] This invention can be used in polymerase chain reaction (PCR).

Claims

1. A PCR method, characterized in that, include: The steps to prepare a PCR container with the following configuration are as follows: substrate, The flow channel is located on one surface of the aforementioned substrate. A pair of filters are located at both ends of the aforementioned flow channel. A pair of air vents are located on the other side of the substrate and are connected to the flow channel via the filter. A thermal circulation region is formed between the pair of filters in the aforementioned flow channel. This thermal circulation region includes a region that causes thermal deformation of the sample, a region that causes the sample to stretch and anneal, and a connecting region linking these regions. The bifurcation point is formed between the pair of filters in the aforementioned flow channel. The bifurcation channel has one end connected to the aforementioned bifurcation point. The sample inlet is located on the other side of the substrate and communicates with the bifurcated flow channel. A flow channel sealing film is adhered to one surface of the substrate to seal the flow channel. A sample inlet sealing film is adhered to the other side of the substrate to seal the sample inlet. An air vent sealing film is attached to the other side of the substrate to seal the air vent. The steps for introducing the sample for PCR from the sample inlet described above; The step of placing the PCR container in a PCR apparatus, wherein the PCR apparatus includes: a temperature control unit including a heater for heating a portion of the flow channel; a pump system including a pump capable of being connected to the air vent; and a fluorescence detector for detecting fluorescence emitted from a sample within the flow channel. The step of connecting the above-mentioned pump system to the above-mentioned air vent; The step of heating the thermally deformed region and the extended / annealed region in the flow channel using the temperature control unit described above. The step of detecting fluorescence emitted from the sample within the aforementioned flow channel; and The pump system is controlled based on the detected changes in fluorescence levels to control the pressure within the flow channel, thereby causing the sample to move back and forth repeatedly between the thermally denatured region and the extended / annealed region to perform PCR.

2. The PCR method as described in claim 1, characterized in that, The steps of introducing a sample into the PCR container include: peeling off the sealing film of the sample inlet, introducing the sample into the sample inlet, and then reattaching the sealing film of the sample inlet.

3. The PCR method as described in claim 1, characterized in that, The steps of introducing a sample into the PCR container include: peeling off the air vent sealing film and the sample inlet sealing film, introducing the sample into the sample inlet, and then reattaching the sample inlet sealing film.

4. The PCR method as described in claim 1, characterized in that, The aforementioned substrate has a filter placement space inside it. The aforementioned filters are housed within the aforementioned filter installation space.

5. The PCR method according to any one of claims 1 to 4, characterized in that, The PCR container described above includes a buffer channel region connected to the sample inlet. The step of introducing a sample for PCR from the above-mentioned sample inlet includes: introducing the sample from the above-mentioned sample inlet into the above-mentioned buffer channel region. Prior to the above-described step of performing PCR on the sample, the step further includes: moving the sample within the buffer channel region to include it within the thermal cycling region, thereby setting it to an initial state.

6. The PCR method as described in claim 5, characterized in that, In the above-described steps of performing PCR on the sample, there is some sample that has not been used for PCR remaining between the sample inlet and the buffer channel area.

7. The PCR method according to any one of claims 1 to 4, characterized in that, The fluorescence detector described above detects fluorescence emitted from the sample within the aforementioned connecting region.

8. A PCR device, characterized in that, The above-mentioned PCR device has the following features: A PCR container disposed within a PCR apparatus includes: a substrate; a flow channel disposed on one surface of the substrate; a pair of filters disposed at both ends of the flow channel; a pair of air vents disposed on the other surface of the substrate and communicating with the flow channel through the filters; a thermal cycling region formed between the pair of filters in the flow channel, the thermal cycling region including a region for thermal denaturation of the sample, a region for stretching and annealing of the sample, and a connecting region connecting these regions; a bifurcation point formed between the pair of filters in the flow channel; a bifurcation flow channel, one end of which is connected to the bifurcation point; a sample inlet disposed on the other surface of the substrate and communicating with the bifurcation flow channel; a flow channel sealing film adhered to the one surface of the substrate for sealing the flow channel; a sample inlet sealing film adhered to the other surface of the substrate for sealing the sample inlet; and an air vent sealing film adhered to the other surface of the substrate for sealing the air vents. A temperature control unit, comprising a heater, for heating the regions in the flow channel where thermal deformation occurs and the regions where extension and annealing occur; A pump system comprising a pump and a nozzle connecting the pump to the air inlet, capable of changing the pressure within the flow channel; as well as A fluorescence detector used to detect fluorescence emitted from the sample within the aforementioned flow channel. By controlling the pump system based on the changes in fluorescence detected by the fluorescence detector, thereby controlling the pressure within the flow channel, the sample is repeatedly moved back and forth between the region where thermal denaturation occurs and the region where extension and annealing occur, thereby performing PCR on the sample.

9. The PCR apparatus as described in claim 8, characterized in that, The aforementioned substrate has a filter placement space inside it. The aforementioned filters are housed within the aforementioned filter installation space.

10. The PCR apparatus as described in claim 8 or 9, characterized in that, The PCR container includes a buffer channel region connected to the sample inlet. During PCR of the sample, unused sample remains between the sample inlet and the buffer channel region.

11. The PCR apparatus as described in claim 8 or 9, characterized in that, The fluorescence detector described above detects fluorescence emitted from the sample within the aforementioned connecting region.

12. A PCR container, characterized in that, include: substrate, The flow channel is located on one surface of the aforementioned substrate. A pair of filters are located at both ends of the aforementioned flow channel. A pair of air vents are located on the other side of the substrate and are connected to the flow channel via the filter. A thermal circulation region is formed between the pair of filters in the aforementioned flow channel. This thermal circulation region includes a region that causes thermal deformation of the sample, a region that causes the sample to stretch and anneal, and a connecting region linking these regions. The bifurcation point is formed between the pair of filters in the aforementioned flow channel. The bifurcation channel has one end connected to the aforementioned bifurcation point. The sample inlet is located on the other side of the substrate and communicates with the bifurcated flow channel. A flow channel sealing film is adhered to one surface of the substrate to seal the flow channel. A sample inlet sealing film is adhered to the other side of the substrate to seal the sample inlet. An air vent sealing film is attached to the other side of the substrate to seal the air vent.

13. The PCR container as described in claim 12, characterized in that, The aforementioned substrate has a filter placement space inside it. The aforementioned filters are housed within the aforementioned filter installation space.

14. The PCR container as described in claim 12 or 13, wherein, The PCR container described above includes a buffer channel area connected to the sample inlet.

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