PCR reaction vessel and PCR apparatus

Abstract

To prevent contamination of a PCR reaction vessel.SOLUTION: A PCR reaction vessel 10 comprises: a substrate 14; a flow channel 12 formed on the substrate 14; a pair of first filter 28 and second filter 30 provided on both ends of the flow channel 12; a pair of first air communication port 24 and second air communication port 26 which communicate with the flow channel 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 channel 12; a branch point 112c formed between the first filter 28 and the second filter 30 in the flow channel 12; a branch flow channel 131 whose one end is connected to the branch point 112c; and a sample introduction port 133 formed on the other end of the branch flow channel 131.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a PCR reaction vessel used in polymerase chain reaction (PCR), a PCR apparatus using the PCR reaction vessel, and a PCR method.

Background Art

[0002] Gene testing is widely used in tests in various medical fields, identification of agricultural crops and pathogenic microorganisms, safety evaluation of foods, and further testing of pathogenic viruses and various infectious diseases. In order to detect a very small amount of DNA, which is a gene, with high sensitivity, a method of analyzing a product obtained by amplifying a part of DNA is known. Among them, the PCR method is a remarkable technique for selectively amplifying a certain part of DNA in a very small amount collected from a living body or the like. In the PCR method, a predetermined thermal cycle is applied to a sample obtained by mixing a biological sample containing DNA and a PCR reagent composed of primers, enzymes, etc., and reactions such as denaturation, annealing, and extension are repeatedly caused to selectively amplify a specific part of DNA.

[0003] In the PCR method, it is common to carry out the reaction by putting a predetermined amount of a target sample into a reaction vessel such as a PCR tube or a microplate (microwell) having a plurality of holes formed therein. In recent years, it has been put into practical use to carry out the reaction using a reaction vessel (also called a chip) having a fine flow path formed on a substrate. In any of the reaction vessels, due to various technological advancements, a predetermined thermal cycle can be given at high speed and with high accuracy in the reaction vessel.

[0004] Patent Document 1 discloses a reaction vessel with a channel formed therein for performing PCR. In this reaction vessel, a channel is formed between two overlapping resin substrates, and a sample inlet for introducing a sample into the channel and a sample outlet for discharging a sample are secured by through holes formed in the resin substrates. A temperature control unit, such as a Peltier element, is arranged in a recess on the back surface of the resin substrate. A nozzle is placed at the sample inlet of the reaction vessel, and the sample can be moved through the channel by supplying and sucking air through the nozzle. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2009-232700 [Overview of the project] [Problems that the invention aims to solve]

[0006] In PCR, contamination from external sources into the system during sample processing must be avoided. If the contamination includes biological fragments other than the target sample, there is a possibility that the DNA contained in these fragments will be amplified. In this case, it will be impossible to accurately perform subsequent analysis using the target sample. Therefore, after the sample is introduced into the reaction vessel, measures must be taken to prevent contamination.

[0007] However, in the invention disclosed in Patent Document 1, if, for example, biological fragments other than the material being processed are attached to the nozzle or the pump that supplies air to the nozzle, there is a risk that these biological fragments may enter the reaction vessel through the sample inlet and cause contamination. Furthermore, it is not practical from a cost and environmental perspective to dispose of the nozzle, pump tip parts, attachments, etc., after each PCR process.

[0008] The present invention has been made in view of these circumstances, and its object is to provide a PCR reaction vessel that can suitably prevent contamination, a PCR apparatus using the PCR reaction vessel, and a PCR method. [Means for solving the problem]

[0009] To solve the above problems, a PCR reaction vessel according to one aspect of the present invention comprises a substrate, a channel formed in the substrate, a pair of filters provided at both ends of the channel, a pair of air vents communicating with the channel through the filters, a thermal cycle region formed between the pair of filters in the channel, a branching point formed between the pair of filters in the channel, a branched channel having one end connected to the branching point, and a sample inlet formed at the other end of the branched channel.

[0010] Another aspect of the present invention is also a PCR reaction vessel. This PCR reaction vessel comprises a substrate, a channel formed in the substrate, a pair of filters provided at both ends of the channel, a pair of air vents communicating with the channel through the filters, a thermal cycle region formed between the pair of filters in the channel, a first branch point formed between the pair of filters in the channel, a first branch channel with one end connected to the first branch point, a first sample inlet formed at the other end of the first branch channel, a second branch point formed between the pair of filters in the channel, a second branch channel with one end connected to the second branch point, and a second sample inlet formed at the other end of the second branch channel.

[0011] The PCR reaction vessel described above may further include a buffer channel region formed between the first and second branching points in the channel.

[0012] The buffer channel region may be set to a predetermined volume corresponding to the amount of sample to be subjected to PCR processing.

[0013] The thermal cycle region may include meandering channels. The thermal cycle region may comprise a pair of reaction regions, each containing a meandering channel, and a connecting region connecting the pair of reaction regions.

[0014] The device may further include sealing films for sealing the air vents and sample inlets.

[0015] The sealing film may be formed to be perforated with a needle.

[0016] Another aspect of the present invention is a PCR apparatus. This apparatus may include the PCR reaction vessel described above, a temperature control unit for adjusting the temperature of the thermal cycle region, and a pump system for controlling the pressure in the flow path via an air vent for moving a sample within the thermal cycle region.

[0017] The pump system may include an air pump of a type that equalizes the pressure on the primary and secondary sides when stopped.

[0018] The air pump may be equipped with a nozzle having a hollow needle at its tip.

[0019] The PCR apparatus may further include a fluorescence detector for detecting fluorescence generated from the sample in the flow path.

[0020] The PCR apparatus may further include a control unit for controlling the pump system based on values ​​detected by a fluorescence detector.

[0021] A further aspect of the present invention is a PCR method. This method comprises the steps of: preparing a PCR reaction vessel comprising a substrate, a channel formed in the substrate, a pair of filters provided at both ends of the channel, a pair of air vents communicating with the channel through the filters, a thermal cycle region formed between the pair of filters in the channel, a branching point formed between the pair of filters in the channel, a branched channel having one end connected to the branching point, and a sample inlet formed at the other end of the branched channel; introducing a sample into the PCR reaction vessel through the sample inlet; setting the PCR reaction vessel in a PCR apparatus equipped with a pump; connecting the nozzle of the pump to the air vents; and moving the sample within the thermal cycle region by controlling the pressure in the channel with the pump.

[0022] During the step of moving the sample, samples that are not to be subjected to PCR may remain in the branched channel.

[0023] Another aspect of the present invention is also a PCR method. This PCR method comprises the steps of: preparing a PCR reaction vessel comprising a substrate, a channel formed in the substrate, a pair of filters provided at both ends of the channel, a pair of air vents communicating with the channel through the filters, a thermal cycle region formed between the pair of filters in the channel, a first branch point formed between the pair of filters in the channel, a first branch channel with one end connected to the first branch point, a first sample inlet formed at the other end of the first branch channel, a second branch point formed between the pair of filters in the channel, a second branch channel with one end connected to the second branch point, and a second sample inlet formed at the other end of the second branch channel; introducing a sample into the PCR reaction vessel through the first sample inlet or the second sample inlet; setting the PCR reaction vessel in a PCR apparatus equipped with a pump; connecting the nozzle of the pump to the air vent; and moving the sample within the thermal cycle region by controlling the pressure in the channel with the pump.

[0024] The PCR reaction vessel further includes a buffer channel region formed between the first branch point and the second branch point in the flow channel, and the above-described PCR method may further include a step of dispensing a sample using the buffer channel region.

[0025] In the step of moving the sample, a sample not used for PCR may remain in the first branch channel and the second branch channel.

Advantages of the Invention

[0026] According to the present invention, it is possible to provide a PCR reaction vessel capable of suitably preventing contamination, a PCR apparatus using the PCR reaction vessel, and a PCR method.

Brief Description of the Drawings

[0027] [Figure 1] FIGS. 1(a) and (b) are diagrams for explaining a PCR reaction vessel according to a first embodiment of the present invention. [Figure 2] It is a cross-sectional view taken along line A-A of the PCR reaction vessel shown in FIG. 1(a). [Figure 3] It is a cross-sectional view taken along line B-B of the PCR reaction vessel shown in FIG. 1(a). [Figure 4] It is a plan view of a substrate included in the PCR reaction vessel according to the first embodiment. [Figure 5] It is a conceptual diagram for explaining the configuration of the PCR reaction vessel according to the first embodiment. [Figure 6] In the first embodiment, it is a diagram schematically showing a state where a sample is introduced into the PCR reaction vessel. [Figure 7] In the first embodiment, it is a diagram showing a state where the third sealing film is pasted back on the substrate again. [Figure 8] It is a diagram for explaining a PCR apparatus using the PCR reaction vessel according to the first embodiment. [Figure 9] In the first embodiment, it is a diagram for explaining a state where the PCR reaction vessel is set at a predetermined position of the PCR apparatus. [Figure 10]This figure shows the connection between the nozzle of the pump system and the air port of the PCR reaction vessel in the first embodiment. [Figure 11] Figure 10 is a cross-sectional view of the PCR reaction vessel at CC. [Figure 12] This figure shows the pump system being operated to move the sample in the first embodiment. [Figure 13] Figures 13(a) and (b) illustrate a PCR reaction vessel according to a second embodiment of the present invention. [Figure 14] Figure 13(a) is a cross-sectional view of the PCR reaction vessel shown in Figure 13(a). [Figure 15] Figure 13(a) is a cross-sectional view of the BB of the PCR reaction vessel. [Figure 16] This is a plan view of the substrate included in the PCR reaction vessel according to the second embodiment. [Figure 17] This is a conceptual diagram illustrating the configuration of the PCR reaction vessel according to the second embodiment. [Figure 18] This figure schematically shows how the sample is introduced into the PCR reaction vessel in the second embodiment. [Figure 19] This figure shows the state in which the third sealing film has been reattached to the substrate in the second embodiment. [Figure 20] This is a diagram illustrating a PCR apparatus using a PCR reaction vessel according to the second embodiment. [Figure 21] This diagram illustrates the state in which the PCR reaction vessel is set in a predetermined position on the PCR apparatus in the second embodiment. [Figure 22] This figure shows the connection between the nozzle of the pump system and the air port of the PCR reaction vessel in the second embodiment. [Figure 23] Figure 22 is a cross-sectional view of the PCR reaction vessel at CC. [Figure 24] This figure shows the pump system being operated to move the sample in the second embodiment. [Modes for carrying out the invention]

[0028] The following describes a PCR reaction vessel and PCR apparatus according to embodiments of the present invention. The same or equivalent components, members, and processes shown in each drawing are denoted by the same reference numerals, and redundant descriptions are omitted as appropriate. Furthermore, the embodiments are illustrative and not limiting to the invention, and not all features or combinations thereof described in the embodiments are necessarily essential to the invention.

[0029] [First Embodiment] Figures 1(a) and 1(b) are diagrams illustrating a PCR reaction vessel 10 according to a first embodiment of the present invention. Figure 1(a) is a plan view of the PCR reaction vessel 10, and Figure 1(b) is a front view of the PCR reaction vessel 10. Figure 2 is a cross-sectional view AA of the PCR reaction vessel 10 shown in Figure 1(a). Figure 3 is a cross-sectional view BB of the PCR reaction vessel 10 shown in Figure 1(a). Figure 4 is a plan view of the substrate 14 provided in the PCR reaction vessel 10. Figure 5 is a conceptual diagram illustrating the configuration of the PCR reaction vessel 10.

[0030] The PCR reaction vessel 10 consists of a resin substrate 14 with groove-shaped channels 12 formed on its lower surface 14a, a channel sealing film 16 attached to the lower surface 14a of the substrate 14 to seal the channels 12, and three sealing films (first sealing film 18, second sealing film 20, and third sealing film 22) attached to the upper surface 14b of the substrate 14.

[0031] The substrate 14 is preferably formed from a material that has good thermal conductivity, is stable against temperature changes, and is resistant to the sample solution used. Furthermore, the substrate 14 is preferably formed from a material that has good moldability, good transparency and barrier properties, and low autofluorescence. Suitable materials for this purpose include inorganic materials such as glass and silicon, as well as resins such as acrylic, polyester, and silicone, with cycloolefin being particularly preferred. An example of the dimensions of the substrate 14 is 70 mm on the long side, 42 mm on the short side, and 3 mm in thickness. An example of the dimensions of the channel 12 formed on the lower surface 14a of the substrate 14 is 0.5 mm in width and 0.5 mm in depth.

[0032] As described above, a groove-shaped channel 12 is formed on the lower surface 14a of the substrate 14, and this channel 12 is sealed by a channel sealing film 16 (see Figure 2). A first air communication port 24 is formed at one end 12a of the channel 12 on the substrate 14. A second air communication port 26 is formed at the other end 12b of the channel 12 on the substrate 14. The pair of first air communication ports 24 and second air communication ports 26 are formed to be exposed on the upper surface 14b of the substrate 14. Such a substrate can be manufactured by injection molding or machining using an NC machine.

[0033] A first filter 28 is provided between a first air vent 24 in the substrate 14 and one end 12a of the flow path 12 (see Figure 2). A second filter 30 is provided between a second air vent 26 in the substrate 14 and the other end 12b of the flow path 12. The pair of first filters 28 and second filters 30 provided at both ends of the flow path 12 have good low-impurity characteristics and allow only air to pass through, preventing contamination so as not to degrade the quality of DNA amplified by PCR. Suitable filter materials include polyethylene and PTFE, which may be porous or hydrophobic. The dimensions of the first filter 28 and second filter 30 are formed so as to fit snugly into the filter installation space formed in the substrate 14.

[0034] A branch channel 131 is formed on the substrate 14 at the branching point 112c between the first filter 28 and the second filter 30, branching off from the channel 12. A sample inlet 133 is formed at the end 31a of the branch channel 131 on the substrate 14 (see Figure 3). The sample inlet 133 is formed to be exposed on the upper surface 14b of the substrate 14.

[0035] The portion of the flow path 12 between the first filter 28 and the branch point 112c forms a thermal cycling region 12e, which includes a high-temperature region and a medium-temperature region, in order to provide a thermal cycle to the sample. The thermal cycling region 12e of the flow path 12 includes a meandering flow path. This is to efficiently transfer the amount of heat supplied from the PCR device during the PCR process to the sample and to ensure that the volume of sample that can be subjected to PCR is above a certain amount. In this first embodiment, a branch point 112c is provided between the thermal cycling region 12e and the second filter 30. However, since the branch point 112c is for introducing the sample to be subjected to PCR into the flow path through the branched flow path 131 and the sample inlet 133 connected to it, there is no functional problem if it is formed between the first filter 28 and the second filter 30. The PCR reaction vessel 10 is to be installed in a PCR device to apply a thermal cycle to the sample and measure optical properties such as fluorescence emitted from the sample. Therefore, the arrangement of each element, including the flow path and branching points, can be arbitrarily selected, taking into consideration the placement of the temperature control unit and fluorescence detection probe described later. In this first embodiment, the branching point 112c is placed closer to the second filter 30, and the thermal cycle region is provided between the branching point 112c and the first filter 28. As a result, the distance in the flow path between the branching point 112c and the first filter 28 can be made relatively large, creating space for the thermal cycle region and also for efficiently arranging the temperature control unit when installed in a PCR device. Conversely, if the branching point 112c is placed closer to the first filter 28, it is more rational to form the thermal cycle region 12e between the branching point 112c and the second filter 30.

[0036] In the PCR reaction vessel 10 according to this first embodiment, most of the channel 12 is formed as a groove exposed on the lower surface 14a of the substrate 14. This is to allow for easy molding by injection molding using a mold or the like. To utilize this groove as a channel, a channel sealing film 16 is attached to the lower surface 14a of the substrate 14. The channel sealing film 16 may have adhesive properties on one main surface, or a functional layer that exhibits adhesiveness or bonding properties when pressed may be formed on one main surface, and it has the function of easily adhering to and integrating with the lower surface 14a of the substrate 14. It is desirable that the channel sealing film 16 be formed from a material having low autofluorescence, including the adhesive. In this respect, transparent films made of cycloolefin polymer, polyester, polypropylene, polyethylene, or acrylic resins are suitable, but are not limited to these. In addition, the channel sealing film 16 may be formed from plate-shaped glass or resin. In this case, rigidity can be expected, which helps to prevent warping and deformation of the PCR reaction vessel 10.

[0037] Furthermore, in the PCR reaction vessel 10 according to this first embodiment, the first air port 24, the second air port 26, the first filter 28, the second filter 30, and the sample inlet 133 are exposed on the upper surface 14b of the substrate 14. Therefore, a first sealing film 18 is attached to the upper surface 14b of the substrate 14 to seal the first air port 24 and the first filter 28. A second sealing film 20 is attached to the upper surface 14b of the substrate 14 to seal the second air port 26 and the second filter 30. A third sealing film 22 is attached to the upper surface 14b of the substrate 14 to seal the sample inlet 133.

[0038] The first sealing film 18 is sized to seal both the first air port 24 and the first filter 28, and the second sealing film 20 is sized to seal both the second air port 26 and the second filter 30 simultaneously. The pressurized pump (described later) is connected to the first air port 24 and the second air port 26 by puncturing the first air port 24 and the second air port 26 with a hollow needle (a pointed hypodermic needle) attached to the tip of the pump. Therefore, the first sealing film 18 and the second sealing film 20 are preferably made of a material and thickness that makes puncture by the needle easy. In this first embodiment, a sealing film sized to seal both the corresponding air ports and filters simultaneously has been described, but they may also be sealed separately. Furthermore, a sealing film that can seal the first air port 24, the first filter 28, the second air port 26, and the second filter 30 all at once (in a single film) may also be used.

[0039] The third sealing film 22 is of a size that can seal the sample inlet 133. To introduce the sample into the flow path 12 through the sample inlet 133, the third sealing film 22 is temporarily peeled off the substrate 14. After introducing a predetermined amount of sample, the third sealing film 22 is returned to the upper surface 14b of the substrate 14 and reattached. Therefore, the third sealing film 22 is preferably a film with adhesive properties that can withstand several cycles of attachment and removal. Alternatively, the third sealing film 22 may be configured to be replaced with a new film after sample introduction; in this case, the importance of attachment / removal properties may be reduced.

[0040] Furthermore, when introducing a sample, it is necessary to temporarily remove either the first sealing film 18 or the second sealing film 20, similar to the third sealing film 22. This is because the sample will not enter the flow path unless an air outlet is created. For this reason, it is desirable that the first sealing film 18 and the second sealing film 20 be films with adhesive properties that can withstand several cycles of application and removal. Alternatively, a new film may be applied after the sample has been introduced.

[0041] The first sealing film 18, the second sealing film 20, and the third sealing film 22 may, like the channel sealing film 16, have an adhesive layer formed on one of their main surfaces, or a functional layer that exhibits tackiness or adhesion upon pressure. It is desirable that the first sealing film 18, the second sealing film 20, and the third sealing film 22 be made from a material having low autofluorescence, including the adhesive. In this respect, transparent films made of resins such as cycloolefin, polyester, polypropylene, polyethylene, or acrylic are suitable, but are not limited to these. Furthermore, as mentioned above, it is desirable that the properties such as tackiness do not deteriorate to the extent that they affect use even after multiple application / removal cycles. However, if the film is peeled off, a sample is introduced, and then a new film is applied, the importance of these application / removal properties may be reduced.

[0042] Next, the method of using the PCR reaction vessel 10 configured as described above will be explained. First, prepare the sample to be amplified by thermal cycling. Examples of samples include a mixture containing two or more types of DNA, to which multiple types of primers, a heat-resistant enzyme, and four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP) are added as PCR reagents. Next, peel off the first sealing film 18 and the third sealing film 22 from the substrate 14, and open the first air port 24 and the sample inlet 133. If the first sealing film 18 is sized to seal both the first air port 24 and the first filter 28 simultaneously, the first sealing film 18 may be completely peeled off the substrate 14, leaving the first air port 24 and the first filter 28 exposed to the atmosphere. However, by not completely peeling off the first sealing film 18 from the substrate 14 and only opening the first air port 24, the first filter 28 is not exposed to the atmosphere, which is effective in preventing contamination. Similarly, when a sealing film is used that can seal the first air vent 24 and the first filter 28 separately, the first filter 28 is not exposed to the atmosphere, which is effective in preventing contamination.

[0043] Next, the sample is introduced into the sample inlet 133 using a dropper, syringe, or the like. Figure 6 schematically shows the sample 70 being introduced into the PCR reaction vessel 10. Note that in Figure 6, the sample 70 is represented by a solid line thicker than the flow path 12 to emphasize its position. It should be noted that this does not represent the sample 70 protruding from the flow path.

[0044] As shown in Figure 6, the sample 70 introduced into the sample inlet 133 is either pushed in by a dropper or syringe, or fills the flow path by capillary action. The sample 70 fills the flow path 12 beyond the branching point 112c in the direction of the thermal cycle region 12e (the direction of the first air port 24). However, the sample 70 does not fill beyond the branching point 112c in the direction of the second air port 26. This is because the second air port 26 is sealed and has no escape route for air.

[0045] Next, as shown in Figure 7, the first sealing film 18 and the third sealing film 22 are reattached to the substrate 14, sealing the first air vent 24 and the sample inlet 133. As described above, new first sealing film 18 and third sealing film 22 may be attached. This completes the introduction of the sample 70 into the PCR reaction vessel 10.

[0046] Figure 8 is a diagram illustrating the PCR apparatus 100 using the PCR reaction vessel 10. Figure 9 is a diagram illustrating the state in which the PCR reaction vessel 10 is set in a predetermined position on the PCR apparatus 100.

[0047] The PCR apparatus 100 includes a fluorescence detection optical probe 122, a first heater 134, and a second heater 135. As shown in Figure 9, the PCR reaction vessel 10 is installed in the PCR apparatus 100 such that the two reaction regions of the thermal cycle region 12e of the flow path 12 are positioned on the first heater 134 and the second heater 135, respectively, and the fluorescence detection optical probe 122 is positioned in the connecting region between the two reaction regions.

[0048] The PCR apparatus 100 further includes a pump system 110 for reciprocating the sample 70 within the thermal cycle region 12e. This pump system 110 comprises a first nozzle 101, a second nozzle 102, a first pump 103, a second pump 104, a first driver 105, a second driver 106, and a control unit 107. The first nozzle 101 of the pump system 110 is connected to the first air port 24 of the PCR reaction vessel 10, and the second nozzle 102 of the pump system 110 is connected to the second air port 26 of the PCR reaction vessel 10. The specific method of connecting the nozzles and air ports will be described later. The pump system 110 moves the sample within the thermal cycle region 12e by controlling the pressure in the flow path 12 via the first air port 24 and the second air port 26.

[0049] 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 supplies heat to individually control the temperature of two reaction regions within the thermal cycle region 12e, and has an area that covers the area of ​​each reaction region. Each heater may also be a resistive heater or a Peltier element, or other means or configuration. 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 page within the thermal cycle region 12e of the flow path 12 at a constant 94°C. The second heater 135 is controlled by the second heater driver 132 to maintain the temperature of the reaction region on the left side of the page at a constant 60°C. The temperature of each reaction region may be measured by a temperature sensor (not shown), such as a thermocouple, and the output to each heater may be controlled by each driver based on the 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 adjusting the temperature of the thermal cycle region 12e, and may include other elements that improve temperature controllability. Hereinafter, the reaction region in the flow path 12 with an ambient temperature of 94°C will be referred to as the "high temperature section 111," and the reaction region in the flow path 12 with an ambient temperature of 60°C will be referred to as the "medium temperature section 112." In this embodiment, a PCR apparatus equipped with a PCR reaction vessel and temperature control unit having a thermal cycle region that sets two temperature levels as two reaction regions will be described in detail, but a PCR apparatus equipped with a PCR reaction vessel and temperature control unit having a thermal cycle region that can set three or more temperature levels may also be used. In this case (not shown in the figure), as an example, a PCR apparatus equipped with a PCR reaction vessel and temperature control unit having reaction regions arranged from left to right as a low temperature section, a medium temperature section, and a high temperature section may be used. In such cases, 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.

[0050] As described above, the pump system 110 is arranged to reciprocate the sample 70 within the thermal cycle region 12e of the flow path 12. The control unit 107 operates the first pump 103 and the second pump 104 alternately under certain conditions via the first driver 105 and the second driver 106, thereby allowing the sample 70 to reciprocate between the high-temperature section 111 and the medium-temperature section 112 of the flow path 12, and providing the sample 70 with a thermal cycle under certain conditions. In the PCR apparatus 100 according to this first embodiment, the first pump 103 and the second pump 104 are both air pumps or blower pumps of a type that, when stopped, instantly equalize the pressure on the primary and secondary sides, and when both are stopped, equalize the pressure on the primary and secondary sides. If a pump of this type is not used, that is, if a pump that maintains the pressure immediately before stopping is used, the sample may continue to move slightly even when the pump stops, preventing the sample from stopping in the predetermined reaction region and making it impossible to properly control the sample temperature. On the other hand, in the PCR apparatus 100 according to this first embodiment, when stopped (open), the external air and the flow path of the PCR reaction vessel are in atmospheric pressure communication and become equal to atmospheric pressure. However, since a filter is provided between the air inlet and the flow path, contamination into the flow path can be prevented.

[0051] Sample 70 can undergo PCR by the thermal cycle described above, but fluorescence from the sample 70 in the flow channel can be detected, and its value can be used as an indicator to determine the progress of PCR or the termination of the reaction. As the fluorescence detection optical probe 122 and driver 121, the FLE-510 optical fiber fluorescence detector manufactured by Nippon Sheet Glass Co., Ltd. can be used, which has a very compact optical system, can measure quickly, and can detect fluorescence regardless of light or dark atmosphere. This optical fiber fluorescence detector can be easily placed even in the narrow space between the two reaction regions within the thermal cycle area. The excitation light / fluorescence wavelength characteristics of this optical fiber fluorescence detector can be tuned to suit the fluorescence characteristics of the sample 70, making it possible to provide an optimal optical and detection system for samples with various characteristics. In addition, multiple fluorescence detection optical probes 122 and drivers 121 may be installed at multiple locations throughout the thermal cycle area 12e. For example, they may be installed to detect fluorescence from the sample 70 in the flow channel in the high-temperature section 111 or the medium-temperature section 112. In addition to functions such as obtaining information to determine the progress and completion of PCR, it can also function as a position sensor to reliably detect whether or not the sample 70 is in the high-temperature section 111 or the medium-temperature section 112.

[0052] 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 probe 122, the first heater driver 130, and the second heater driver 132 are controlled by the CPU 141 to operate optimally. Furthermore, if the apparatus has a reaction region with three temperature levels as described above, a third heater driver (not shown) is also controlled by the CPU in addition to the above.

[0053] Figure 10 shows the connection between the nozzle of the pump system and the air port of the PCR reaction vessel. Figure 11 is a cross-sectional view of the PCR reaction vessel 10 shown in Figure 10. As described above, the first nozzle 101 is connected to the first air port 24, and the second nozzle 102 is connected to the second air port 26.

[0054] As shown in Figure 11, a hollow needle 150 is provided at the tip of the first nozzle 101. By perforating the first sealing film 18 with this needle 150, the first nozzle 101 is connected to the first air vent 24. The connection between the second nozzle 102 and the second air vent 26 is similar.

[0055] The needle 150 is provided with a packing 151 made of a flexible resin that adheres tightly to the surface of the sealing film in order to ensure airtightness around the connection point. Immediately after the PCR reaction vessel 10 is set in the PCR device 100, the pump system 110 is not operating and is open to the atmosphere, so the pressure inside the flow path is equal to atmospheric pressure.

[0056] Figure 12 shows the pump system 110 being operated to move the sample 70. Either the first pump 103 or the second pump 104 is operated to move the sample 70 to the high-temperature section 111 or the medium-temperature section 112 of the thermal cycle region 12e. In Figure 12, the second pump 104, to which the second nozzle 102 is connected, is operated, and the first pump 103, to which the first nozzle 101 is connected, is stopped. That is, the first air port 24, to which the first nozzle 101 extending from the first pump 103 is connected, is open to atmospheric pressure. When the second pump 104 is operated and air is sent from the second nozzle 102 to the second air port 26, the sample 70 moves, passing through the medium-temperature section 112 and moving to the high-temperature section 111. This state is considered the initial state.

[0057] More specifically, at the same time as or immediately before the start of operation of the second pump 104, the fluorescence emitted from the sample in the flow path is monitored using the fluorescence detection optical probe 122. When there is nothing at the measurement point of the fluorescence detection optical probe 122, the detected fluorescence is zero or at the background level, but when the sample 70 is present at the measurement point, fluorescence is detected. Therefore, by starting the fluorescence monitoring before the start of operation of the second pump 104, and when the fluorescence value rises from the background level and then falls back to the background level, it is recognized that the sample 70 has completed its movement to the high-temperature section 111, and the operation of the second pump 104 is stopped at this point, completing the initial setting. Furthermore, if the fluorescence detection optical probe is also in the high-temperature section 111, the sample 70 can be stopped in the high-temperature section 111 more reliably.

[0058] Note that the sample 70 located within the branched channel 131 will remain in that location even when the second pump 104 is operated. This is because the sample inlet 133 is sealed with the third sealing film 22. The sample 70 located within this branched channel 131 will not be subjected to PCR.

[0059] After setting up to the initial state, the sample 70 is subjected to thermal cycling to allow PCR to proceed. Fluorescence measurement using the fluorescence detection optical probe 122 is continued.

[0060] (A) First, the sample 70 is placed in the high-temperature section 111 (at an atmosphere of approximately 94°C) for 1 to 30 seconds (Deneturation: thermal denaturation process). This process denatures the double-stranded DNA into single-stranded DNA.

[0061] (B) Next, the first pump 103, to which the first nozzle 101 is connected, is operated to move the sample 70 to the medium temperature section 112 (approximately 60°C atmosphere). Specifically, the sample 70 is pushed from the high temperature section 111 towards the medium temperature section 112 by the action of the first pump 103. Since fluorescence measurement by the fluorescence detection optical probe 122 is continuing, the first pump 103 is stopped when the fluorescence level rises from the background level as the sample 70 passes the measurement point of the fluorescence detection optical probe 122 and then falls again (or after a certain period of time has elapsed since the fluorescence level fell). Furthermore, if the fluorescence detection optical probe 122 is in the medium temperature section 112, the sample 70 can be stopped in the medium temperature section 112 more reliably.

[0062] (C) In the medium temperature section 112, the sample 70 is left to stand for 3 to 60 seconds (Annealing + Elongation). This process causes the primers that were previously contained in the sample 70 to bind and further elongate the DNA.

[0063] (D) Next, the second pump 104, to which the second nozzle 102 is connected, is activated to move the sample 70 from the medium temperature section 112 to the high temperature section 111. The timing for stopping the pump operation is determined, as above, from the change in the amount of fluorescence measured by the fluorescence detection optical probe 122. After moving the sample 70 to the high temperature section 111, it is left to wait for 1 to 30 seconds to allow it to undergo thermal denaturation.

[0064] (E) Steps (B) to (D) above are repeated for a predetermined number of cycles to thermal cycle the sample 70, and the DNA contained in the sample 70 undergoes multiple cycles of thermal denaturation-annealing-extension to amplify the DNA. The number of cycles is determined appropriately depending on the combination of target DNA, primers, enzymes, etc.

[0065] After a predetermined number of thermal cycles are completed, the first pump 103 and the second pump 104 are stopped, and the PCR is terminated. Even when a predetermined number of thermal cycles are being performed, fluorescence is measured by the fluorescence detection optical probe 122, and as the DNA contained in the sample 70 is amplified, the fluorescence detected from the sample 70 increases. This allows for accurate determination of the concentration of the sample 70.

[0066] According to the PCR reaction vessel 10 of the first embodiment, contamination in the flow path 12 can be prevented by providing a first filter 28 between the first air port 24 and the flow path 12, and a second filter 30 between the second air port 26 and the flow path 12. Implementing contamination prevention measures on the pump system 110 side tends to be costly, but in the PCR reaction vessel 10 of this first embodiment, contamination can be prevented solely on the PCR reaction vessel 10 side, making it economical. Furthermore, when the PCR reaction vessel is used as disposable, the filters are always new, further reducing the cost of contamination prevention. In addition, regarding the disposal of the PCR reaction vessel, the sample is largely sealed within the PCR reaction vessel, which is beneficial from a safety and environmental perspective.

[0067] In the PCR apparatus 100 according to this first embodiment, the sample can be moved back and forth within the flow path 12 of the PCR reaction vessel 10 by alternately operating the first pump 103 and the second pump 104, which equalize the pressure on the primary and secondary sides when stopped. In this case, excessive pressure is not applied to the sample during liquid delivery (pressure is applied to the sample in the flow path), and furthermore, the pressure in the flow path is not reduced, thus preventing evaporation or boiling (foaming) of the liquid containing the sample due to the action of the high-temperature section 111.

[0068] Furthermore, in the PCR apparatus 100 according to this first embodiment, fluorescence from the sample is constantly monitored in the thermal cycling region even during PCR (real-time PCR). This allows the timing of the end of PCR to be determined based on the measured fluorescence amount. In addition, by monitoring the change in fluorescence with the fluorescence detection optical probe 122, the passage of the sample can be known, and the alternating operation of the first pump 103 and the second pump 104 can be controlled based on the change in fluorescence amount accompanying the passage, so that the sample to be subjected to PCR can be accurately positioned in the high-temperature section 111 or the medium-temperature section 112 of the thermal cycling region.

[0069] On the other hand, in the case of a PCR reaction vessel and PCR apparatus equipped with three temperature-controlled reaction regions—a high-temperature section, a medium-temperature section, and a low-temperature section—as described above, it becomes possible to have the high-temperature section handle thermal denaturation, the medium-temperature section handle annealing, and the low-temperature section handle extension. The control of these processes can also be easily developed and improved by those skilled in the art based on the detailed explanation above. Furthermore, whether to have two or three temperature levels in the reaction region can be appropriately selected by those skilled in the art depending on the characteristics of the sample.

[0070] [Second Embodiment] Figures 13(a) and (b) are diagrams illustrating a PCR reaction vessel 210 according to a second embodiment of the present invention. Figure 13(a) is a plan view of the PCR reaction vessel 210, and Figure 13(b) is a front view of the PCR reaction vessel 210. Figure 14 is a cross-sectional view AA of the PCR reaction vessel 210 shown in Figure 13(a). Figure 15 is a cross-sectional view BB of the PCR reaction vessel 210 shown in Figure 13(a). Figure 16 is a plan view of the substrate 214 provided in the PCR reaction vessel 210. Figure 17 is a conceptual diagram illustrating the configuration of the PCR reaction vessel 210. The PCR reaction vessel 210 in the second embodiment differs from the first embodiment in that it has two branching points (first branching point 212c and second branching point 212d), branched channels extending from there, and two sample inlets (first branching channel 231 and first sample inlet 233, second branching channel 232 and second sample inlet 234), and also has a buffer channel region 212f between the first branching point 212c and the second branching point 212d.

[0071] The PCR reaction vessel 210 consists of a resin substrate 214 with groove-shaped channels 212 formed on its lower surface 214a, a channel sealing film 216 attached to the lower surface 214a of the substrate 214 to seal the channels 212, and three sealing films (first sealing film 218, second sealing film 220, and third sealing film 222) attached to the upper surface 214b of the substrate 214.

[0072] The substrate 214 is preferably formed from a material that has good thermal conductivity, is stable against temperature changes, and is resistant to the sample solution used. Furthermore, the substrate 214 is preferably formed from a material that has good moldability, good transparency and barrier properties, and low autofluorescence. Suitable materials for this purpose include inorganic materials such as glass and silicon, as well as resins such as acrylic, polyester, and silicone, with cycloolefin being particularly preferred. An example of the dimensions of the substrate 214 is 70 mm on the long side, 42 mm on the short side, and 3 mm in thickness. An example of the dimensions of the channel 212 formed on the lower surface 214a of the substrate 214 is 0.5 mm in width and 0.5 mm in depth.

[0073] As described above, a groove-shaped channel 212 is formed on the lower surface 214a of the substrate 214, and this channel 212 is sealed by a channel sealing film 216 (see Figure 14). A first air communication port 224 is formed at one end 212a of the channel 212 on the substrate 214. A second air communication port 226 is formed at the other end 212b of the channel 212 on the substrate 214. The pair of first air communication ports 224 and second air communication ports 226 are formed to be exposed on the upper surface 214b of the substrate 214. Such a substrate can be manufactured by injection molding or cutting using an NC machine.

[0074] A first filter 228 is provided between a first air inlet 224 in the substrate 214 and one end 212a of the flow path 212 (see Figure 14). A second filter 230 is provided between a second air inlet 226 in the substrate 214 and the other end 212b of the flow path 212. The pair of first filters 228 and second filters 230 provided at both ends of the flow path 212 have good low-impurity characteristics and allow only air to pass through, preventing contamination so as not to degrade the quality of DNA amplified by PCR. Suitable filter materials include polyethylene and PTFE, which may be porous or hydrophobic. The dimensions of the first filter 228 and second filter 230 are formed so as to fit snugly into the filter installation space formed in the substrate 214.

[0075] A first branch channel 231 is formed on the substrate 214 at the first branching point 212c between the first filter 228 and the second filter 230, branching off from the channel 212. A first sample inlet 233 is formed at the end 231a of the first branch channel 231 on the substrate 214 (see Figure 15). A second branch channel 232 is further formed on the substrate 214 at the second branching point 212d between the first branching point 212c and the second filter 230, branching off from the channel 212. A second sample inlet 234 is provided at the end 232a of the second branch channel 232 on the substrate 214. The first sample inlet 233 and the second sample inlet 234 are formed to be exposed on the upper surface 214b of the substrate 214.

[0076] The portion of the channel 212 between the first filter 228 and the first branch point 212c forms a thermal cycling region 212e, which includes a high-temperature region and a medium-temperature region, in order to provide a thermal cycle to the sample. The thermal cycling region 212e of the channel 212 includes a meandering channel. This is to efficiently transfer the heat supplied from the PCR instrument during the PCR process to the sample and to ensure that the volume of sample that can be subjected to PCR is above a certain amount. The thermal cycling region 212e comprises a pair of reaction regions, each containing a meandering channel, and a connecting region that connects the pair of reaction regions.

[0077] The portion of the channel 212 between the first branching point 212c and the second branching point 212d forms a buffer channel region 212f. The buffer channel region 212f of channel 212 includes a meandering channel. The volume of the buffer channel region 212f of channel 212 is set to a predetermined volume corresponding to the amount of sample to be subjected to PCR processing. The function of the buffer channel region will be described later.

[0078] In the PCR reaction vessel 210 according to this second embodiment, most of the channel 212 is formed as a groove exposed on the lower surface 214a of the substrate 214. This is to allow for easy molding by injection molding using a mold or the like. To utilize this groove as a channel, a channel sealing film 216 is attached to the lower surface 214a of the substrate 214. The channel sealing film 216 may have adhesive properties on one main surface, or a functional layer that exhibits adhesiveness or bonding properties upon pressure may be formed on one main surface, providing the function of easily adhering to and integrating with the lower surface 214a of the substrate 214. It is desirable that the channel sealing film 216 be formed from a material having low autofluorescence, including the adhesive. In this respect, transparent films made of cycloolefin polymer, polyester, polypropylene, polyethylene, or acrylic resins are suitable, but are not limited to these. The channel sealing film 216 may also be formed from plate-shaped glass or resin. In this case, rigidity can be expected, which helps prevent warping and deformation of the PCR reaction vessel 210.

[0079] Furthermore, in the PCR reaction vessel 210 according to this second embodiment, the first air port 224, the second air port 226, the first sample inlet 233, the second sample inlet 234, the first filter 228, and the second filter 230 are exposed on the upper surface 214b of the substrate 214. Therefore, a first sealing film 218 is attached to the upper surface 214b of the substrate 214 to seal the first air port 224 and the first filter 228. Also, a second sealing film 220 is attached to the upper surface 214b of the substrate 214 to seal the second air port 226 and the second filter 230. Also, a third sealing film 222 is attached to the upper surface 214b of the substrate 14 to seal the first sample inlet 233 and the second sample inlet 234.

[0080] The first sealing film 218 is sized to seal both the first air port 224 and the first filter 228, while the second sealing film 220 is sized to seal both the second air port 226 and the second filter 230 simultaneously. The pressurized pump (described later) is connected to the first air port 224 and the second air port 226 by puncturing the first air port 224 and the second air port 226 with a hollow needle (a pointed hypodermic needle) attached to the tip of the pump. Therefore, the first sealing film 218 and the second sealing film 220 are preferably made of a material and thickness that makes puncture by the needle easy. In this second embodiment, a sealing film sized to seal both the corresponding air ports and filters simultaneously has been described, but they may also be sealed separately. Furthermore, a sealing film capable of sealing the first air port 224, the first filter 228, the second air port 226, and the second filter 230 all at once (in a single film) may also be used.

[0081] The third sealing film 222 is sized to simultaneously seal the first sample inlet 233 and the second sample inlet 234. To introduce the sample into the flow path 212 through the first sample inlet 233 and the second sample inlet 234, the third sealing film 222 is temporarily peeled off the substrate 214. After introducing a predetermined amount of sample, the third sealing film 222 is returned to the upper surface 214b of the substrate 214 and reattached. Therefore, the third sealing film 222 is preferably a film with adhesive properties that can withstand several cycles of attachment and removal. Alternatively, the third sealing film 222 may be configured to be replaced with a new film after sample introduction; in this case, the importance of attachment / removal properties may be reduced. Furthermore, while this second embodiment describes a sealing film sized to simultaneously seal the first sample inlet 233 and the second sample inlet 234, they may also be sealed separately.

[0082] The first sealing film 218, the second sealing film 220, and the third sealing film 222 may, like the channel sealing film 216, have an adhesive layer formed on one main surface, or a functional layer that exhibits tackiness or adhesion upon pressure. It is desirable that the first sealing film 218, the second sealing film 220, and the third sealing film 222 be made from a material having low autofluorescence, including the adhesive. In this respect, transparent films made of resins such as cycloolefin (COP), polyester, polypropylene, polyethylene, or acrylic are suitable, but are not limited to these. Furthermore, as mentioned above, it is desirable that the properties such as tackiness do not deteriorate to the extent that they affect use even after multiple application / removal cycles. However, if a new film is applied after peeling off and introducing a sample, the importance of these application / removal properties may be reduced.

[0083] Next, the method of using the PCR reaction vessel 210 configured as described above will be explained. First, prepare the sample to be amplified by thermal cycling. Examples of samples include a mixture containing two or more types of DNA, to which multiple types of primers, a heat-resistant enzyme, and four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP) are added as PCR reagents. Next, peel off the third sealing film 222 from the substrate 214 and open the first sample inlet 233 and the second sample inlet 234.

[0084] Next, the sample is introduced into either the first sample inlet 233 or the second sample inlet 234 using a dropper, syringe, or the like. Figure 18 schematically shows the sample 270 being introduced into the PCR reaction vessel 210. Note that in Figure 18, the sample 270 is represented by a solid line thicker than the flow path 212 to emphasize its position. It should be noted that this does not represent the sample 270 protruding from the flow path.

[0085] As shown in Figure 18, the sample 270 introduced into either the first sample inlet 233 or the second sample inlet 234 is pushed in by a dropper or syringe, or fills the channel by capillary action. The sample 270 fills the buffer channel region 212f between the first branching point 212c and the second branching point 212d in the channel 212. However, the sample 270 does not penetrate beyond the first branching point 212c and the second branching point 212d at both ends of the buffer channel region into the thermal cycle region 212e or the second air communication port 26 of the channel 212. This is because both ends of the channel (i.e., the first air communication port 224 and the second air communication port 226) are sealed at this point, and there is no escape for the air.

[0086] Next, as shown in Figure 19, the third sealing film 222 is reattached to the substrate 214 to seal the first sample inlet 233 and the second sample inlet 234. A new third sealing film 222 may be applied as described above. This completes the introduction of the sample 270 into the PCR reaction vessel 210.

[0087] Figure 20 is a diagram illustrating the PCR apparatus 300 using the PCR reaction vessel 210. Figure 21 is a diagram illustrating the state in which the PCR reaction vessel 210 is set in a predetermined position on the PCR apparatus 300.

[0088] The PCR apparatus 300 includes a fluorescence detection optical probe 2122, a first heater 2134, and a second heater 2135. As shown in Figure 21, the PCR reaction vessel 210 is installed in the PCR apparatus 300 such that a pair of reaction regions of the thermal cycle region 212e of the flow path 212 are positioned on the first heater 2134 and the second heater 2135, and the fluorescence detection optical probe 2122 is positioned in the connecting region between the pair of reaction regions. The PCR apparatus 300 can utilize the PCR apparatus applied in the PCR reaction vessel according to the first embodiment.

[0089] The PCR apparatus 300 further includes a pump system 2110 for reciprocating the sample 270 within the thermal cycle region 212e. This pump system 2110 comprises a first nozzle 2101, a second nozzle 2102, a first pump 2103, a second pump 2104, a first driver 2105, a second driver 2106, and a control unit 2107. The first nozzle 2101 of the pump system 2110 is connected to the first air port 224 of the PCR reaction vessel 210, and the second nozzle 2102 of the PCR reaction vessel 210 is connected to the second air port 226 of the PCR reaction vessel 210. The specific method of connecting the nozzles and air ports will be described later. The pump system 2110 moves the sample within the thermal cycle region 212e by controlling the pressure in the flow path 212 via the first air port 224 and the second air port 226.

[0090] 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 supplies heat to individually control the temperature of a pair of reaction regions within the thermal cycle region 212e, and may use means or configurations such as resistance heating or Peltier elements. For example, the first heater 2134 is controlled by the first heater driver 2130 to maintain the temperature of the reaction region on the right side of the page within the thermal cycle region 212e of the flow path 212 at a constant 94°C. The second heater 2135 is controlled by the second heater driver 2132 to maintain the temperature of the reaction region on the left side of the page at a constant 60°C. The temperature of each reaction region may be measured by a temperature sensor (not shown), such as a thermocouple, and the output to each heater may be controlled by each driver based on the 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 control unit for adjusting the temperature of the thermal cycle region 212e, and may include other elements that improve temperature controllability. This temperature control unit allows the thermal cycle region 212e of the flow path 212 to be divided into two regions with different ambient temperatures. A temperature sensor (not shown), such as a thermocouple, for measuring the temperature at the vicinity of each heater may be included, and other configurations that improve temperature controllability may also be included. Hereinafter, the reaction region in the flow path 212 with an ambient temperature of 94°C will be referred to as the "high-temperature region 2111," and the reaction region in the flow path 212 with an ambient temperature of 60°C will be referred to as the "medium-temperature region 2112." In this embodiment, a PCR apparatus comprising a PCR reaction vessel and temperature control unit having a thermal cycle region that sets two temperature levels as two reaction regions will be described in detail. However, a PCR apparatus comprising a PCR reaction vessel and temperature control unit having a thermal cycle region that can set three or more temperature levels may also be used. In this case (not shown in the figure), for example, a PCR apparatus comprising a PCR reaction vessel and temperature control unit having reaction regions arranged from left to right as a low temperature section, a medium temperature section, and a high temperature section may be used. In such a case, for example, the low temperature section is controlled to be maintained at 50-70°C, the medium temperature section at 72°C, and the high temperature section at 94°C.

[0091] As described above, the pump system 2110 is positioned to reciprocate the sample 270 within the thermal cycle region 212e of the flow path 212. The control unit 2107 operates the first pump 2103 and the second pump 2104 alternately under certain conditions via the first driver 2105 and the second driver 2106, thereby allowing the sample 270 to reciprocate between the high-temperature section 2111 and the medium-temperature section 2112 of the flow path 212, and providing the sample 270 with a thermal cycle under certain conditions. In the PCR apparatus 300 according to this second embodiment, the first pump 2103 and the second pump 2104 are both air pumps or blower pumps of a type that, when stopped, instantly equalize the pressure on the primary and secondary sides, and when both are stopped, equalize the pressure on the primary and secondary sides. If a pump of this type is not used, that is, if a pump that maintains the pressure immediately before stopping is used, even when the pump is stopped, the sample may continue to move slightly, preventing the sample from stopping in the predetermined reaction region and making it impossible to properly control the sample temperature. On the other hand, when the system is stopped (open), the external air and the flow path of the PCR reaction vessel are in atmospheric pressure communication, and the pressure becomes equal to atmospheric pressure. However, because a filter is provided between the air inlet and the flow path, contamination into the flow path can be prevented.

[0092] Sample 270 can undergo PCR using the thermal cycle described above, but fluorescence from the sample 270 in the flow channel can be detected, and its value can be used as an indicator to determine the progress of PCR or the termination of the reaction. As the fluorescence detection optical probe 2122 and driver 2121, the FLE-510 optical fiber fluorescence detector manufactured by Nippon Sheet Glass Co., Ltd. can be used, which has a very compact optical system, can measure quickly, and can detect fluorescence regardless of the light or dark atmosphere. This optical fiber fluorescence detector can be easily placed even in the narrow space between two temperature regions within the thermal cycle area. The excitation light / fluorescence wavelength characteristics of this optical fiber fluorescence detector can be tuned to suit the fluorescence characteristics of sample 270, making it possible to provide an optimal optical and detection system for samples with various characteristics. In addition, the thermal cycle area 212e may be equipped with multiple fluorescence detection optical probes 2122 and drivers 2121 installed at various locations. For example, they may be installed to detect fluorescence from the sample 270 in the flow channel in the high-temperature section 2111 or the medium-temperature section 2112. In addition to functions such as obtaining information to determine the progress and completion of PCR, it can also function as a position sensor to reliably detect whether or not the sample 270 is in the high-temperature section 2111 or the medium-temperature section 2112.

[0093] 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 probe 2122, the first heater driver 2130, and the second heater driver 2132 are controlled by the CPU 2141 to operate optimally. Furthermore, if the apparatus has a reaction region with three temperature levels as described above, a third heater driver (not shown) is also controlled by the CPU in addition to the above.

[0094] Figure 22 shows the connection between the nozzle of the pump system and the air port of the PCR reaction vessel. Figure 23 is a cross-sectional view of the PCR reaction vessel 210 shown in Figure 23. As described above, the first nozzle 2101 is connected to the first air port 224, and the second nozzle 2102 is connected to the second air port 226.

[0095] As shown in Figure 23, a needle 2150 is provided at the tip of the first nozzle 2101. By perforating the first sealing film 218 with this needle 2150, the first nozzle 2101 is connected to the first air vent 224. The connection between the second nozzle 2102 and the second air vent 226 is similar.

[0096] The needle 2150 is equipped with a packing 2151 made of a flexible resin that adheres tightly to the surface of the sealing film in order to ensure airtightness around the connection point. Immediately after the PCR reaction vessel 210 is set in the PCR device 300, the pump system 2110 is not operating and is open to the atmosphere, so the pressure inside the flow path is equal to atmospheric pressure.

[0097] Figure 24 shows the pump system 2110 being 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 channel region 212f of the flow path 212 to the high-temperature section 2111 or the medium-temperature section 2112 of the thermal cycle region 212e. In Figure 24, the second pump 2104, to which the second nozzle 2102 is connected, is operated, and the first pump 2103, to which the first nozzle 2101 is connected, is stopped. That is, the first air communication port 224, to which the first nozzle 2101 extending from the first pump 2103 is connected, is open to atmospheric pressure. When the second pump 2104 is activated and air is sent from the second nozzle 2102 to the second air communication port 226, the sample 270 moves out of the buffer channel region 212f of the channel 212, passes through the medium temperature section 2112, and moves to the high temperature section 2111. This state is considered the initial state.

[0098] More specifically, at the same time as or immediately before the start of operation of the second pump 2104, the fluorescence detection optical probe 2122 is used to start monitoring the fluorescence emitted from the flow path. When there is nothing at the measurement point of the fluorescence detection optical probe 2122, the detected fluorescence is zero or at the background level, but when the sample 270 is present at the measurement point, fluorescence is detected. Therefore, by starting the fluorescence monitoring from the start of operation of the second pump 2104, and when the fluorescence value rises from the background level and then falls back to the background level, it is recognized that the sample 270 has completed its movement to the high-temperature section 2111, and at this point the operation of the second pump 2104 is stopped, completing the initial setting. Furthermore, if the fluorescence detection optical probe 2122 is also located in the high-temperature section 2111, it is possible to more reliably stop the sample 270 in the high-temperature section 2111.

[0099] It should be noted that the sample 270 located in the first branch channel 231 and the second branch channel 232 remains in place even when the second pump 2104 is operated. This is because the first sample inlet 233 and the second sample inlet 234 are sealed with the third sealing film 222. The sample 270 located in the first branch channel 231 and the second branch channel 232 is not subjected to PCR. Therefore, even if there is variation in the amount of sample initially introduced into the PCR reaction vessel 210, by setting the volume of the buffer channel region 212f of the channel 212 formed in the PCR reaction vessel 210 to a predetermined volume corresponding to the amount of sample to be subjected to PCR, a desired constant amount of sample can always be delivered to the thermal cycle region 212e of the channel 212, and the amount of fluorescence that affects the progress and termination of PCR can be kept approximately constant. In other words, the buffer channel region 212f of the channel 212 has a dispensing function that can extract a desired constant amount of sample.

[0100] After setting up to the initial state, the sample 270 is subjected to thermal cycling to allow PCR to proceed. Fluorescence measurement using the fluorescence detection optical probe 2122 is continued.

[0101] (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: thermal denaturation process). This process denatures the double-stranded DNA into single-stranded DNA.

[0102] (B) Next, the first pump 2103, to which the first nozzle 2101 is connected, is operated to move the sample 270 to the medium temperature section 2112 (approximately 60°C atmosphere). Specifically, the sample 270 is pushed from the high temperature section 2111 towards the medium temperature section 2112 by the action of the first pump 2103. Since fluorescence measurement by the fluorescence detection optical probe 2122 is continuing, the first pump 2103 is stopped when the fluorescence level rises from the background level as the sample 270 passes the measurement point of the fluorescence detection optical probe 2122 and then falls again (or after a certain period of time has elapsed since the fluorescence level fell). If the fluorescence detection optical probe 2122 is in the medium temperature section 2112, the sample 270 can be stopped in the medium temperature section 2112 more reliably.

[0103] (C) In the medium temperature section 2112, the sample 270 is left to stand for 3 to 60 seconds (Annealing + Elongation). This process allows the primers that were previously contained in the sample 270 to bind, resulting in further elongated DNA.

[0104] (D) Next, the second pump 2104, to which the second nozzle 2102 is connected, is activated to move the sample 270 from the medium temperature section 2112 to the high temperature section 2111. The timing for stopping the pump operation is determined, as above, from the change in the amount of fluorescence measured by the fluorescence detection optical probe 2122. After moving the sample 270 to the high temperature section 2111, it is left to wait for 1 to 30 seconds to allow it to undergo thermal denaturation.

[0105] (E) Steps (B) to (D) above are repeated for a predetermined number of cycles to thermal cycle the sample 270, and the DNA contained in the sample 270 undergoes multiple cycles of thermal denaturation, annealing, and extension, thereby amplifying the DNA. The number of cycles is determined appropriately depending on the combination of target DNA, primers, enzymes, etc.

[0106] After a predetermined number of thermal cycles are completed, the first pump 2103 and the second pump 2104 are stopped, and the PCR is terminated. Even when a predetermined number of thermal cycles are being performed, fluorescence is measured by the fluorescence detection optical probe 122, and as the DNA contained in the sample 270 is amplified, the fluorescence detected from the sample 270 increases. This allows for the accurate determination of the concentration of the sample 270.

[0107] According to the PCR reaction vessel 210 of this second embodiment, a first filter 228 is provided between the first air port 224 and the flow path 212, and a second filter 230 is provided between the second air port 226 and the flow path 212, thereby preventing contamination within the flow path 212. Implementing contamination prevention measures on the pump system 2110 side tends to be costly, but in the PCR reaction vessel 210 of this second embodiment, contamination can be prevented solely on the PCR reaction vessel 210 side, making it economical. Furthermore, when the PCR reaction vessel is used as disposable, the filters are always new, further reducing the cost of contamination prevention. In addition, regarding the disposal of the PCR reaction vessel, the sample is largely sealed within the PCR reaction vessel, which is beneficial from a safety and environmental perspective.

[0108] Furthermore, according to the PCR reaction vessel 210 of this second embodiment, by providing a buffer channel region in the channel 212, the sample to be used for PCR can be dispensed, and only the required amount of sample can always be sent into the thermal cycle region of the channel 212.

[0109] In the PCR apparatus 300 according to this second embodiment, the sample can be moved back and forth within the flow path 212 of the PCR reaction vessel 210 by alternately operating the first pump 2103 and the second pump 2104, which equalize the pressure on the primary and secondary sides when stopped. In this case, excessive pressure is not applied to the sample during liquid delivery (pressure is applied to the sample in the flow path), and furthermore, the pressure in the flow path is not reduced, thus preventing evaporation or boiling (foaming) of the liquid containing the sample due to the action of the high-temperature section 2111.

[0110] Furthermore, in the PCR apparatus 300 according to this second embodiment, fluorescence from the sample is constantly monitored in the thermal cycling region even during PCR (real-time PCR). This allows the timing of the end of PCR to be determined based on the measured fluorescence amount. In addition, by monitoring the change in fluorescence with the fluorescence detection optical probe 2122, the passage of the sample can be known, and the alternating operation of the first pump 2103 and the second pump 2104 can be controlled based on the change in fluorescence amount accompanying the passage, so that the sample to be subjected to PCR can be accurately positioned in the high-temperature section 2111 or the medium-temperature section 2112 of the thermal cycling region.

[0111] On the other hand, in the case of a PCR reaction vessel and PCR apparatus equipped with three temperature-controlled reaction regions—a high-temperature section, a medium-temperature section, and a low-temperature section—as described above, it becomes possible to have the high-temperature section handle thermal denaturation, the medium-temperature section handle annealing, and the low-temperature section handle extension. The control of these processes can also be easily developed and improved by those skilled in the art based on the detailed explanation above. Furthermore, whether to have two or three temperature levels in the reaction region can be appropriately selected by those skilled in the art depending on the characteristics of the sample.

[0112] The present invention has been described above based on embodiments. These embodiments are illustrative, and it will be understood by those skilled in the art that various modifications are possible in combinations of these components and processing processes, and that such modifications also fall within the scope of the present invention.

[0113] In the above-described embodiment, a pair of pumps are placed at both ends of the flow path so that the primary and secondary pressures are equal when stopped. However, a pump capable of both pressurizing and suction may be provided at only one end of the flow path, with the other end open to atmospheric pressure. That is, the sample is moved within the thermal cycle region by controlling the pressure in the flow path via the first or second air inlet. In this case, the process of switching the operation of the pair of pumps at a fixed timing becomes unnecessary, making pump control easier.

[0114] Furthermore, in the above-described embodiment, the measurement point of the fluorescence detection optical probe was placed midway between the high-temperature and medium-temperature sections. However, the measurement points of the fluorescence detection optical probe may also be placed in the high-temperature and medium-temperature sections respectively. In this case, the positioning accuracy of the sample can be improved. [Explanation of symbols]

[0115] 10, 210 PCR reaction vessel, 12, 212 flow channel, 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 inlet, 26, 226 second air inlet, 28, 228 first filter, 30, 230 second filter, 70, 270 sample, 100, 300 PCR device, 101, 2101 first nozzle, 102, 2102 second nozzle, 103, 2103 first pump, 104, 2104 second pump, 105, 2105 first driver, 106, 2106 second driver, 107, 2107 control unit. 110, 2110 Pump system, 111, 2111 High temperature section, 112, 2112 Medium temperature section, 121, 2121 Driver, 122, 2122 Optical probe for fluorescence detection, 130, 2130 First heater driver, 131 Branch channel, 132, 2132 Second heater driver, 133 Sample inlet, 134, 2134 First heater, 135, 2135 Second heater, 141, 2141 CPU, 231 First branch channel, 232 Second branch channel, 233 First sample inlet, 234 Second sample inlet. [Industrial applicability]

[0116] This invention can be used in polymerase chain reaction (PCR).

Claims

1. circuit board and A channel formed in the substrate, A pair of filters provided at both ends of the aforementioned flow path, A pair of air vents communicating with the flow path through the filter, The aforementioned flow path includes a sample inlet for introducing a sample to be subjected to PCR, A thermal cycle region formed between the pair of filters in the flow path, having a high-temperature section corresponding to the thermal denaturation step of PCR and a medium-temperature section corresponding to the annealing and extension steps, Equipped with, The sample repeatedly moves back and forth between the high-temperature section and the medium-temperature section. The aforementioned substrate is a parallel plate-shaped substrate, The filter has a smaller thickness than the substrate, The PCR reaction vessel is characterized in that the filter fits into a filter installation space formed in the substrate.

2. The PCR reaction vessel according to claim 1, characterized in that the high-temperature section and the medium-temperature section each include a meandering channel.

3. A sealing film for sealing the air vent, the filter, and the sample inlet, A PCR reaction vessel according to claim 1 or 2, further comprising a flow channel sealing film for sealing the high-temperature section and the medium-temperature section in the flow channel.

4. The PCR reaction vessel according to any one of claims 1 to 3, characterized in that the distance between the sample inlet and the medium-temperature section is shorter than the distance between the sample inlet and the high-temperature section.

5. A PCR reaction vessel according to any one of claims 1 to 4, A temperature control unit for adjusting the temperature of the thermal cycle region, A pump system that controls the pressure in the flow path via the air communication port in order to move the sample within the thermal cycle region, Equipped with, The pump system includes a pump connected to the air communication port, The PCR apparatus is characterized in that the pump is such that the pressures on the primary and secondary sides become equal when the pump is stopped.

6. The pump is equipped with a hollow needle, The PCR apparatus according to claim 5, characterized in that the pump system and the flow path are connected by perforating the sealing film that seals the air vent with the needle.

7. The PCR apparatus according to claim 5 or 6, further comprising a fluorescence detector for detecting fluorescence generated from a sample in the aforementioned flow channel.

8. The thermal cycling region includes a connecting region that connects the high-temperature section and the medium-temperature section. The PCR apparatus according to claim 7, characterized in that the fluorescence detector detects fluorescence from the connection region.

9. The system further comprises a control unit for controlling the pump system based on the value detected by the fluorescence detector, The PCR apparatus according to claim 7 or 8, characterized in that the control unit determines the progress of PCR based on the value detected by the fluorescence detector.

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