PCR device and PCR method

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 for both ends of the flow channel 12; a pair of first air communication port 24 and second air communication port 26 which communicates with the flow channel 12 through the first filter 28 and the second filter 30; a thermal cycle region 12e which is formed between the first filter 28 and the second filter 30 in the flow channel 12; a branch point 112c which is 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 a polymerase chain reaction (PCR), a PCR device using the PCR reaction vessel, and a PCR method. [Background technology]

[0002] Genetic testing is widely used in various medical fields, identification of agricultural crops and pathogenic microorganisms, food safety evaluation, and even testing for pathogenic viruses and various infectious diseases. In order to detect minute amounts of DNA, which is a gene, with high sensitivity, a method is known in which a part of DNA is amplified and analyzed. Among them, the PCR method is a notable technology that selectively amplifies a certain part of a very small amount of DNA taken from a living body. In the PCR method, a sample containing a biological sample containing DNA is mixed with PCR reagents consisting of primers and enzymes, and a specified thermal cycle is applied to the sample, causing repeated reactions such as denaturation, annealing, and extension, thereby selectively amplifying a specific part of the DNA.

[0003] In PCR, a predetermined amount of a target sample is generally placed in a reaction vessel such as a PCR tube or a microplate (microwell) with multiple holes, but in recent years, it has become common to use a reaction vessel (also called a chip) equipped with fine flow paths formed on a substrate. In either type of reaction vessel, various technological advances have made it possible to apply a predetermined thermal cycle quickly and accurately within the reaction vessel.

[0004] Patent Document 1 discloses a reaction vessel in which a flow path for PCR is formed. In this reaction vessel, the flow path is formed between two overlapping resin substrates, and a sample inlet for introducing a sample into the flow path and a sample outlet for discharging the sample are secured by through holes formed in the resin substrates. A temperature control unit, for example, made of a Peltier element, is disposed in a recess on the rear surface of the resin substrate. A nozzle is disposed in the sample inlet of the reaction vessel, and the sample can be moved in the flow path by supplying and suctioning air through the nozzle. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2009-232700 A Summary of the Invention [Problem to be solved by the invention]

[0006] In PCR, contamination from the outside into the system must be avoided during sample processing. If contamination includes biological fragments other than the target of processing, the DNA contained in the biological fragments other than the target of processing may be amplified. In this case, the target sample cannot be used for subsequent analysis accurately. 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, for example, if biological fragments other than the target to be processed are attached to the nozzle or the pump that sends air to the nozzle, the biological fragments other than the target to be processed may enter the reaction vessel through the sample inlet, causing contamination. In addition, disposing of the nozzle, the tip parts of the pump, attachments, etc. after each PCR process is not realistic from the viewpoints of cost and environment.

[0008] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a PCR reaction vessel capable of effectively preventing contamination, and a PCR device and a PCR method using the PCR reaction vessel. [Means for solving the problem]

[0009] In order to solve the above problems, a PCR reaction vessel according to one embodiment of the present invention comprises a substrate, a flow path formed on the substrate, a pair of filters provided at both ends of the flow path, a pair of air communication ports communicating with the flow path through the filters, a thermal cycle region formed between the pair of filters in the flow path, a branch point formed between the pair of filters in the flow path, a branch flow path having one end connected to the branch point, and a sample introduction port formed at the other end of the branch flow path.

[0010] Another embodiment of the present invention is also a PCR reaction vessel comprising a substrate, a flow channel formed on the substrate, a pair of filters provided at both ends of the flow channel, a pair of air communication ports communicating with the flow channel through the filters, a thermal cycle 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 having one end 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 having one end connected to the second branch point, and a second sample inlet formed at the other end of the second branch flow channel.

[0011] The above-mentioned PCR reaction vessel may further include a buffer flow channel region formed between the first branch point and the second branch point in the flow channel.

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

[0013] The thermal cycling region may include a serpentine flow path.The thermal cycling region may include a pair of reaction regions, each of which includes a serpentine flow path, and a connection region connecting the pair of reaction regions.

[0014] The device may further include a sealing film for sealing the air communication port and the sample introduction port.

[0015] The sealing film may be formed so as to be pierceable by a needle.

[0016] Another embodiment of the present invention is a PCR device that may include the above-mentioned PCR reaction vessel, a temperature control unit for controlling the temperature of the thermal cycle region, and a pump system for controlling the pressure in the flow channel through an air communication port in order to move a sample in the thermal cycle region.

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

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

[0019] The PCR device may further include a fluorescence detector for detecting fluorescence emitted from the sample in the flow channel.

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

[0021] Yet another aspect of the present invention is a PCR method, which includes the steps of preparing a PCR reaction vessel including a substrate, a flow path formed on the substrate, a pair of filters provided at both ends of the flow path, a pair of air communication ports communicating with the flow path through the filters, a thermal cycle region formed between the pair of filters in the flow path, a branch point formed between the pair of filters in the flow path, a branch flow path having one end connected to the branch point, and a sample introduction port formed at the other end of the branch flow path, introducing a sample into the PCR reaction vessel through the sample introduction port, setting the PCR reaction vessel in a PCR device equipped with a pump, connecting a nozzle of the pump to the air communication port, and controlling the pressure in the flow path with the pump to move the sample in the thermal cycle region.

[0022] In the step of moving the sample, the sample that is not subjected to PCR may remain in the branched channel.

[0023] Yet another aspect of the present invention is also a PCR method, which includes the steps of: preparing a PCR reaction vessel including a substrate, a flow path formed on the substrate, a pair of filters provided at both ends of the flow path, a pair of air communication ports communicating with the flow path through the filters, a thermal cycle region formed between the pair of filters in the flow path, a first branch point formed between the pair of filters in the flow path, a first branch flow path having one end connected to the first branch point, a first sample inlet formed at the other end of the first branch flow path, a second branch point formed between the pair of filters in the flow path, a second branch flow path having one end connected to the second branch point, and a second sample inlet formed at the other end of the second branch flow path, 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 device equipped with a pump, connecting a nozzle of the pump to the air communication port, and controlling the pressure in the flow path with the pump to move the sample in the thermal cycle region.

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

[0025] In the step of moving the sample, the sample that is not subjected to PCR may remain in the first branch channel and the second branch channel. Effect of the Invention

[0026] According to the present invention, it is possible to provide a PCR reaction vessel capable of preferably preventing contamination, and a PCR device and a PCR method using the PCR reaction vessel. [Brief description of the drawings]

[0027] [Figure 1] 1(a) and (b) are diagrams illustrating a PCR reaction vessel according to a first embodiment of the present invention. [Diagram 2] 2 is a cross-sectional view of the PCR reaction vessel shown in FIG. 1(a) taken along line AA. [Diagram 3] BB cross-sectional view of the PCR reaction vessel shown in FIG. [Figure 4] FIG. 2 is a plan view of a substrate provided in the PCR reaction container according to the first embodiment. [Diagram 5] FIG. 2 is a conceptual diagram for explaining the configuration of a PCR reaction container according to the first embodiment. [Figure 6] FIG. 2 is a diagram showing a schematic view of a state in which a sample is introduced into a PCR reaction vessel in the first embodiment. [Figure 7] FIG. 11 is a diagram showing a state in which the third sealing film is attached back to the substrate in the first embodiment. [Figure 8] FIG. 2 is a diagram for explaining a PCR device using a PCR reaction container according to the first embodiment. [Figure 9] FIG. 2 is a diagram for explaining a state in which a PCR reaction container is set at a predetermined position in the PCR device in the first embodiment. [Figure 10]FIG. 11 is a diagram showing a state in which a nozzle of a pump system and an air communication port of a PCR reaction container are connected in the first embodiment. [Figure 11] 11 is a CC cross-sectional view of the PCR reaction vessel shown in FIG. [Figure 12] FIG. 2 is a diagram showing how a pump system is operated to move a sample in the first embodiment. [Figure 13] 13(a) and (b) are diagrams illustrating a PCR reaction vessel according to a second embodiment of the present invention. [Figure 14] 13(a) is a cross-sectional view of the PCR reaction vessel taken along line AA in FIG. [Figure 15] 13(a) is a cross-sectional view taken along the line BB of the PCR reaction vessel shown in FIG. [Figure 16] FIG. 11 is a plan view of a substrate provided in a PCR reaction container according to a second embodiment. [Figure 17] FIG. 11 is a conceptual diagram for explaining the configuration of a PCR reaction container according to a second embodiment. [Figure 18] FIG. 11 is a diagram showing a schematic view of a state in which a sample is introduced into a PCR reaction vessel in the second embodiment. [Figure 19] FIG. 11 is a diagram showing a state in which the third sealing film is attached back to the substrate in the second embodiment. [Figure 20] FIG. 11 is a diagram for explaining a PCR device using a PCR reaction container according to a second embodiment. [Figure 21] FIG. 11 is a diagram for explaining a state in which a PCR reaction container is set at a predetermined position in a PCR device in a second embodiment. [Figure 22] FIG. 11 is a diagram showing a state in which a nozzle of a pump system and an air communication port of a PCR reaction container are connected in the second embodiment. [Diagram 23] 23 is a CC cross-sectional view of the PCR reaction vessel shown in FIG. 22. [Figure 24] FIG. 11 is a diagram showing how a pump system is operated to move a sample in the second embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The following describes a PCR reaction vessel and a PCR device according to an embodiment of the present invention. The same or equivalent components, parts, and processes shown in each drawing are given the same reference numerals, and duplicated descriptions are omitted as appropriate. In addition, the embodiments are illustrative and do not limit the invention, and all the features and combinations thereof described in the embodiments are not necessarily essential to the invention.

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

[0030] The PCR reaction vessel 10 consists of a resin substrate 14 having a groove-shaped flow path 12 formed on its bottom surface 14a, a flow path sealing film 16 for sealing the flow path 12 attached to the bottom surface 14a of the substrate 14, and three sealing films (a first sealing film 18, a second sealing film 20 and a third sealing film 22) attached to the top surface 14b of the substrate 14.

[0031] The substrate 14 is preferably made of a material that has good thermal conductivity, is stable against temperature changes, and is not easily affected by the sample solution used. Furthermore, the substrate 14 is preferably made of a material that has good moldability, good transparency and barrier properties, and low autofluorescence. Such materials include inorganic materials such as glass and silicon, as well as resins such as acrylic, polyester, and silicone, and among these, cycloolefin is preferable. An example of the dimensions of the substrate 14 is a long side of 70 mm, a short side of 42 mm, and a thickness of 3 mm. An example of the dimensions of the flow channel 12 formed on the lower surface 14a of the substrate 14 is a width of 0.5 mm and a depth of 0.5 mm.

[0032] As described above, a groove-shaped flow path 12 is formed in the lower surface 14a of the substrate 14, and this flow path 12 is sealed by a flow path sealing film 16 (see FIG. 2). A first air communication port 24 is formed at the position of one end 12a of the flow path 12 in the substrate 14. A second air communication port 26 is formed at the position of the other end 12b of the flow path 12 in the substrate 14. The pair of first air communication port 24 and second air communication port 26 are formed so as to be exposed on the upper surface 14b of the substrate 14. Such a substrate can be produced by injection molding or cutting processing using an NC processing machine or the like.

[0033] A first filter 28 is provided between the first air communication port 24 in the substrate 14 and one end 12a of the flow path 12 (see FIG. 2). A second filter 30 is provided between the second air communication port 26 in the substrate 14 and the other end 12b of the flow path 12. The pair of first filter 28 and second filter 30 provided at both ends of the flow path 12 have good low impurity characteristics, and also allow only air to pass through, preventing contamination so that the quality of DNA amplified by PCR is not deteriorated. As the filter material, polyethylene, PTFE, etc. are suitable, and may be porous or hydrophobic. The dimensions of the first filter 28 and the second filter 30 are formed to fit snugly into the filter installation space formed in the substrate 14.

[0034] In substrate 14, a branch flow path 131 is formed, which branches off from flow path 12 at branch point 112c between first filter 28 and second filter 30. In substrate 14, a sample introduction port 133 is formed at the position of terminal end 31a of branch flow path 131 (see FIG. 3). Sample introduction port 133 is formed so as to be exposed on upper surface 14b of substrate 14.

[0035] The portion of the flow path 12 between the first filter 28 and the branch point 112c forms a thermal cycle region 12e, which is scheduled to have a high temperature region and a medium temperature region, in order to apply a thermal cycle to the sample. The thermal cycle region 12e of the flow path 12 includes a meandering flow path. This is to efficiently apply the heat provided by the PCR device to the sample in the PCR process and to ensure that the volume of the sample that can be used for PCR is equal to or greater than a certain amount. In this first embodiment, the branch point 112c is provided between the thermal cycle region 12e and the second filter 30, but since the branch point 112c is for introducing the sample to be used for PCR into the flow path through the branch flow path 131 and the sample introduction port 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 intended to be installed in a PCR device, to subject the sample to a thermal cycle, and to measure optical properties such as fluorescence emitted from the sample. Therefore, the arrangement of each element, including the flow path and the branch point, may be selected arbitrarily, taking into consideration the arrangement of a temperature control unit and a fluorescence detection probe, which will be described later. In the first embodiment, the branch point 112c is arranged closer to the second filter 30, and the thermal cycle area is provided between the branch point 112c and the first filter 28. This allows the distance on the flow path between the branch point 112c and the first filter 28 to be relatively large, and when the vessel is installed in the thermal cycle area or in the PCR device, a space is created to efficiently arrange the temperature control unit. Conversely, if the branch point 112c is arranged closer to the first filter 28, it is more reasonable to form the thermal cycle area 12e between the branch point 112c and the second filter 30.

[0036] In the PCR reaction vessel 10 according to the first embodiment, most of the flow channel 12 is formed in a groove shape exposed on the lower surface 14a of the substrate 14. This is to facilitate molding by injection molding using a mold or the like. In order to utilize this groove as a flow channel, a flow channel sealing film 16 is attached to the lower surface 14a of the substrate 14. The flow channel sealing film 16 may have adhesiveness on one main surface, or a functional layer that exerts adhesiveness or adhesion by pressing may be formed on one main surface, and has a function of easily adhering to and integrating with the lower surface 14a of the substrate 14. It is desirable that the flow channel sealing film 16 is formed from a material having low autofluorescence, including the adhesive. In this respect, a transparent film made of a resin such as a cycloolefin polymer, polyester, polypropylene, polyethylene, or acrylic is suitable, but is not limited to these. The flow channel sealing film 16 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 10.

[0037] In the PCR reaction container 10 according to the first embodiment, the first air communication port 24, the second air communication port 26, the first filter 28, the second filter 30, and the sample introduction port 133 are exposed on the upper surface 14b of the substrate 14. Therefore, the first sealing film 18 is attached to the upper surface 14b of the substrate 14 to seal the first air communication port 24 and the first filter 28. In addition, the second sealing film 20 is attached to the upper surface 14b of the substrate 14 to seal the second air communication port 26 and the second filter 30. In addition, the third sealing film 22 is attached to the upper surface 14b of the substrate 14 to seal the sample introduction port 133.

[0038] The first sealing film 18 has a size capable of simultaneously sealing the first air communication port 24 and the first filter 28, and the second sealing film 20 has a size capable of simultaneously sealing the second air communication port 26 and the second filter 30. A pressurizing pump (described later) is connected to the first air communication port 24 and the second air communication port 26 by perforating the first air communication port 24 and the second air communication port 26 with a hollow needle (a syringe needle with a sharp tip) provided at the tip of the pump. For this reason, the first sealing film 18 and the second sealing film 20 are preferably films made of a material and thickness that can be easily perforated by a needle. In the first embodiment, a sealing film of a size capable of simultaneously sealing the corresponding air communication port and the filter has been described, but a mode in which these are sealed separately is also possible. Also, a sealing film capable of sealing the first air communication port 24, the first filter 28, the second air communication port 26, and the second filter 30 together (as one piece) may be used.

[0039] The third sealing film 22 is of a size capable of sealing the sample introduction port 133. The introduction of the sample into the flow channel 12 through the sample introduction port 133 is performed by once peeling the third sealing film 22 from the substrate 14, and after the introduction of a predetermined amount of sample, the third sealing film 22 is returned and attached to the upper surface 14b of the substrate 14 again. For this reason, the third sealing film 22 is preferably a film having an adhesiveness that can withstand several cycles of attachment / detachment. The third sealing film 22 may be of a type in which a new film is attached after the introduction of the sample, in which case the importance of the characteristics relating to attachment / detachment may be alleviated.

[0040] In addition, when introducing a sample, either the first sealing film 18 or the second sealing film 20 must be peeled off once, 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, the first sealing film 18 and the second sealing film 20 are desirably films having adhesiveness that can withstand several cycles of attachment / detachment. Also, a new film may be attached after the introduction of the sample.

[0041] The first sealing film 18, the second sealing film 20, and the third sealing film 22 may have an adhesive layer formed on one main surface, or a functional layer that exerts adhesiveness or adhesion by pressing, similar to the flow path sealing film 16. The first sealing film 18, the second sealing film 20, and the third sealing film 22 are preferably formed from a material having low autofluorescence, including the adhesive. In this respect, a transparent film made of a resin such as cycloolefin, polyester, polypropylene, polyethylene, or acrylic is suitable, but is not limited to these. As described above, it is desirable that the adhesiveness and other properties do not deteriorate to a degree that affects use even after multiple attachment / peeling operations, but in the case of an embodiment in which a new film is attached after peeling and introduction of a sample, the importance of the properties related to attachment / peeling may be mitigated.

[0042] Next, a method of using the PCR reaction vessel 10 configured as above will be described. First, a sample to be amplified by thermal cycling is prepared. The sample may be a mixture containing two or more types of DNA, to which a plurality of types of primers, a heat-resistant enzyme, and four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP) are added as PCR reagents. Next, the first sealing film 18 and the third sealing film 22 are peeled off from the substrate 14, and the first air communication port 24 and the sample introduction port 133 are opened. If the first sealing film 18 is of a size that can simultaneously seal the first air communication port 24 and the first filter 28, the first sealing film 18 may be completely peeled off from the substrate 14 to expose the first air communication port 24 and the first filter 28 to the atmosphere, but by opening only the first air communication port 24 without completely peeling off the first sealing film 18 from the substrate 14, the first filter 28 is not exposed to the atmosphere, which is effective in preventing contamination. Similarly, when a sealing film capable of sealing the first air communication port 24 and the first filter 28 separately is used, the first filter 28 is not exposed to the atmosphere, which is effective in preventing contamination.

[0043] Next, a sample is introduced into the sample introduction port 133 using a dropper, syringe, or the like. Figure 6 shows a schematic diagram of a state in which a sample 70 has been introduced into the PCR reaction vessel 10. Note that in Figure 6, in order to emphasize the position of the sample 70, the sample 70 is represented by a solid line that is thicker than the flow channel 12. Please note that this does not represent a state in which the sample 70 protrudes from the flow channel.

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

[0045] 7, the first sealing film 18 and the third sealing film 22 are attached back to the substrate 14 to seal the first air communication port 24 and the sample introduction port 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 container 10.

[0046] Fig. 8 is a diagram illustrating a PCR device 100 using a PCR reaction container 10. Fig. 9 is a diagram illustrating a state in which the PCR reaction container 10 is set in a predetermined position in the PCR device 100.

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

[0048] The PCR device 100 further includes a pump system 110 for reciprocating the sample 70 in the thermal cycle region 12e. The pump system 110 includes 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 communication port 24 of the PCR reaction vessel 10, and the second nozzle 102 of the pump system 110 is connected to the second air communication port 26 of the PCR reaction vessel 10. A specific method of connecting the nozzles and the air communication port will be described later. The pump system 110 controls the pressure in the flow channel 12 via the first air communication port 24 and the second air communication port 26 to move the sample in the thermal cycle region 12e.

[0049] In the PCR device 100 according to the first embodiment, the first heater 134 and the second heater 135 are set to different temperatures. Each heater provides heat to individually control the temperature of two reaction regions in the thermal cycle region 12e, and has an area that covers the area of ​​each reaction region. Each heater may be a means or configuration such as resistance heating or a Peltier element. For example, the first heater 134 is controlled by the first heater driver 130 so as to maintain the temperature of the reaction region on the right side of the paper in the thermal cycle region 12e of the flow channel 12 at a constant 94°C. The second heater 135 is controlled by the second heater driver 132 so as to maintain the temperature of the reaction region on the left side of the paper 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. In this way, 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 controlling the temperature of the thermal cycle region 12e, and may include other elements that improve the controllability of the temperature. Hereinafter, the reaction region in the flow channel 12 with an ambient temperature of 94°C is referred to as the "high temperature section 111", and the reaction region in the flow channel 12 with an ambient temperature of 60°C is referred to as the "medium temperature section 112". In addition, in this embodiment, a PCR device equipped with a PCR reaction vessel having a thermal cycle region in which two levels of temperature regions are set as two reaction regions and a temperature control section will be described in detail, but a PCR device equipped with a PCR reaction vessel having a thermal cycle region in which three or more levels of temperature regions can be set and a temperature control section may also be used. In this case (not shown), as an example, a PCR device equipped with a PCR reaction vessel having reaction regions arranged as a low temperature section, a medium temperature section, and a high temperature section from the left side of the paper and a temperature control section may be used. In such a case, the low temperature section is controlled to be maintained at 50 to 70°C, the medium temperature section at 72°C, and the high temperature section at 94°C, for example.

[0050] As described above, the pump system 110 is arranged to reciprocate the sample 70 in the thermal cycle region 12e of the flow channel 12. The control unit 107 alternately operates the first pump 103 and the second pump 104 under certain conditions through the first driver 105 and the second driver 106, so that the sample 70 can be reciprocated between the high temperature section 111 and the medium temperature section 112 of the flow channel 12, and the sample 70 can be subjected to a thermal cycle under certain conditions. In the PCR device 100 according to the first embodiment, the first pump 103 and the second pump 104 are air pumps or blower pumps of a type in which the air pressures on the primary side and the secondary side become equal instantly when either of them is stopped, and when either of them is stopped, the air pressures on the primary side and the secondary side become equal. If such a type of pump is not used, that is, if a pump that maintains the pressure immediately before the pump is stopped is used, a phenomenon occurs in which the sample continues to move slightly even when the pump is stopped, and the sample does not stop in the specified reaction region, and the temperature of the sample cannot be controlled appropriately. On the other hand, in the PCR device 100 according to the first embodiment, when the device is stopped (open), the external air and the flow path of the PCR reaction container are air-pressure-equal to atmospheric pressure; however, since a filter is provided between the air communication port and the flow path, contamination of the flow path can be prevented.

[0051] PCR can be performed on the sample 70 by the above-mentioned thermal cycle, and the fluorescence from the sample 70 in the flow path can be detected and used as an index for judging the progress of PCR and the end of the reaction. The optical probe 122 for detecting fluorescence and the driver 121 can be an optical fiber type fluorescence detector FLE-510 manufactured by Nippon Sheet Glass Co., Ltd., which has a very compact optical system, can perform rapid measurement, and can detect fluorescence regardless of the light or dark atmosphere. This optical fiber type fluorescence detector can be easily arranged in the narrow space between the two reaction areas in the thermal cycle area. This optical fiber type fluorescence detector can be tuned to have the wavelength characteristics of its excitation light / fluorescence suitable for the fluorescent characteristics of the sample 70, and can provide an optimal optical / detection system for samples with various characteristics. The optical probe 122 for detecting fluorescence and the driver 121 may be provided 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 path in the high temperature section 111 or the medium temperature section 112. In addition to the function of obtaining information for judging the progress and completion of PCR, it can also function as a position sensor for reliably detecting whether the sample 70 is in the high temperature section 111 or the medium temperature section 112 or not.

[0052] In the PCR device 100 configured as above, the control unit 107 of the pump system 110, the driver 121 of the optical probe 122 for detecting fluorescence, the first heater driver 130 and the second heater driver 132 are controlled so as to operate optimally by the CPU 141. Furthermore, in the case where a reaction region in which three levels of temperature are set as described above is provided, a third heater driver (not shown) is also controlled by the CPU in addition to the above.

[0053] Fig. 10 is a diagram showing a state in which the nozzle of the pump system and the air communication port of the PCR reaction vessel are connected. Fig. 11 is a CC cross-sectional view of the PCR reaction vessel 10 shown in Fig. 10. As described above, the first nozzle 101 is connected to the first air communication port 24, and the second nozzle 102 is connected to the second air communication port 26.

[0054] 11, a hollow needle 150 is provided at the tip of the first nozzle 101. The first nozzle 101 is connected to the first air communication port 24 by piercing the first sealing film 18 with this needle 150. The second nozzle 102 and the second air communication port 26 are connected in a similar manner.

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

[0056] FIG. 12 shows the state in which the pump system 110 is 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 FIG. 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 communication port 24 to which the first nozzle 101 is connected, which extends from the first pump 103, is open to atmospheric pressure. When the second pump 104 is operated to send air from the second nozzle 102 to the second air communication port 26, the sample 70 moves, passes through the medium temperature section 112, and moves to the high temperature section 111. This state is the initial state.

[0057] More specifically, at the same time as or immediately before the start of the operation of the second pump 104, the fluorescence emitted from the sample in the flow path is started to be monitored using the optical probe for detecting fluorescence 122. When there is nothing at the measurement point of the optical probe for detecting fluorescence 122, the detected fluorescence is zero or at the background level, but when the sample 70 is present at the measurement point, the fluorescence is detected. Therefore, the monitoring of the fluorescence is started before the start of the 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 moving to the high temperature section 111, and the operation of the second pump 104 is stopped at this point, thereby completing the setting of the initial state. Furthermore, if the optical probe for detecting fluorescence is located further in the high temperature section 111, the sample 70 can be more reliably stopped in the high temperature section 111.

[0058] It should be noted here that the sample 70 located in the branch flow path 131 remains in place for the most part 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 in this branch flow path 131 is not subjected to PCR.

[0059] After setting the initial state, the sample 70 is subjected to a thermal cycle to allow PCR to proceed. Measurement of fluorescence by the fluorescence detection optical probe 122 continues.

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

[0061] (B) Next, the first pump 103 connected to the first nozzle 101 is operated to move the sample 70 to the medium temperature section 112 (atmosphere of about 60° C.). Specifically, the sample 70 is pushed from the high temperature section 111 toward the medium temperature section 112 by the action of the first pump 103. Since the fluorescence measurement by the fluorescence detection optical probe 122 continues, the operation of the first pump 103 is stopped when the amount of fluorescence 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 when a certain time has passed since the amount of fluorescence fell). Also, if the fluorescence detection optical probe 122 is in the medium temperature section 112, the sample 70 can be more reliably stopped in the medium temperature section 112.

[0062] (C) In the medium temperature section 112, the sample 70 is left waiting for 3 to 60 seconds (Annealing+Elongation: Annealing step+Elongation step). Through this step, the primers that were previously contained in the sample 70 are bound to each other, resulting in further elongated DNA.

[0063] (D) Next, the second pump 104 connected to the second nozzle 102 is operated to move the sample 70 from the medium temperature section 112 to the high temperature section 111. The timing to stop the pump operation is determined from the fluctuation in the amount of fluorescence measured by the fluorescence detection optical probe 122, as described above. After the sample 70 is moved to the high temperature section 111, it is left to stand for 1 to 30 seconds to be thermally denatured.

[0064] (E) The above steps (B) to (D) are repeated a predetermined number of cycles to subject the sample 70 to thermal cycling, and the DNA contained in the sample 70 is subjected to multiple cycles of thermal denaturation-annealing-extension steps, thereby amplifying the DNA. The number of cycles is appropriately determined depending on the combination of the target DNA, primers, enzymes, etc.

[0065] After the predetermined number of thermal cycles are completed, the first pump 103 and the second pump 104 are stopped to terminate the PCR. Even when the predetermined number of thermal cycles are being applied, the 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 makes it possible to accurately know the concentration of the sample 70.

[0066] According to the PCR reaction vessel 10 of the first embodiment, the first filter 28 is provided between the first air communication port 24 and the flow path 12, and the second filter 30 is provided between the second air communication port 26 and the flow path 12, thereby preventing contamination in the flow path 12. Although taking measures to prevent contamination on the pump system 110 side tends to be costly, the PCR reaction vessel 10 of the first embodiment can prevent contamination only on the PCR reaction vessel 10 side, which is economical. Furthermore, when the PCR reaction vessel is used as a disposable vessel, the filter is always new, so that contamination can be further prevented at low cost. Furthermore, with regard to disposal of the PCR reaction vessel, since the sample is substantially sealed in the PCR reaction vessel, it is also meaningful in terms of safety and the environment.

[0067] In the PCR device 100 according to the first embodiment, the first pump 103 and the second pump 104, whose primary and secondary pressures are equal when stopped, are alternately operated to move the sample back and forth within the flow path 12 of the PCR reaction vessel 10. In this case, since excessive pressure is not applied to the sample during liquid transfer (pressure is applied to the sample in the flow path), and pressure within the flow path is not reduced, evaporation and boiling (foaming) of the liquid containing the sample due to the action of the high-temperature section 111 can be prevented.

[0068] Furthermore, in the PCR device 100 according to the first embodiment, the fluorescence from the sample is constantly monitored in the thermal cycle region even during PCR (real-time PCR). This allows the end timing of PCR to be determined based on the measured amount of fluorescence. Furthermore, the passage of the sample can be detected by monitoring the change in fluorescence using the fluorescence detection optical probe 122, and the alternating operations of the first pump 103 and the second pump 104 can be controlled based on the change in the amount of fluorescence 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 cycle region.

[0069] On the other hand, in the case of a PCR reaction vessel and a PCR device having a reaction region with three temperature levels controlled, namely, a high temperature region, a medium temperature region, and a low temperature region, it becomes possible to carry out each step of thermal denaturation in the high temperature region, annealing in the medium temperature region, and extension in the low temperature region, and the control of these steps can be easily developed and improved by a person skilled in the art based on the above detailed explanation. In addition, whether the reaction region has two or three levels can be appropriately selected by a person skilled in the art depending on the characteristics of the sample.

[0070] [Second embodiment] 13(a) and (b) are diagrams for explaining a PCR reaction vessel 210 according to a second embodiment of the present invention. FIG. 13(a) is a plan view of the PCR reaction vessel 210, and FIG. 13(b) is a front view of the PCR reaction vessel 210. FIG. 14 is an AA cross-sectional view of the PCR reaction vessel 210 shown in FIG. 13(a). FIG. 15 is a BB cross-sectional view of the PCR reaction vessel 210 shown in FIG. 13(a). FIG. 16 is a plan view of a substrate 214 provided in the PCR reaction vessel 210. FIG. 17 is a conceptual diagram for explaining the configuration of the PCR reaction vessel 210. The PCR reaction vessel 210 in the second embodiment has two branch points (a first branch point 212c and a second branch point 212d) and two branch flow paths and sample introduction ports extending from them (a first branch flow path 231 and a first sample introduction port 233, and a second branch flow path 232 and a second sample introduction port 234), and differs from the first embodiment in that it has a buffer flow path region 212f between the first branch point 212c and the second branch point 212d.

[0071] The PCR reaction vessel 210 consists of a resin substrate 214 having a groove-shaped flow path 212 formed on its lower surface 214a, a flow path sealing film 216 for sealing the flow path 212, which is attached to the lower surface 214a of the substrate 214, 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 upper surface 214b of the substrate 214.

[0072] The substrate 214 is preferably made of a material that has good thermal conductivity, is stable against temperature changes, and is not easily affected by the sample solution used. Furthermore, the substrate 214 is preferably made of a material that has good moldability, good transparency and barrier properties, and low autofluorescence. Such materials include inorganic materials such as glass and silicon, as well as resins such as acrylic, polyester, and silicone, and among these, cycloolefin is preferable. An example of the dimensions of the substrate 214 is a long side of 70 mm, a short side of 42 mm, and a thickness of 3 mm. An example of the dimensions of the flow channel 212 formed on the lower surface 214a of the substrate 214 is a width of 0.5 mm and a depth of 0.5 mm.

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

[0074] A first filter 228 is provided between the first air communication port 224 in the substrate 214 and one end 212a of the flow path 212 (see FIG. 14). A second filter 230 is provided between the second air communication port 226 in the substrate 214 and the other end 212b of the flow path 212. The pair of first filter 228 and second filter 230 provided at both ends of the flow path 212 have good low impurity characteristics, and also pass only air, preventing contamination so that the quality of DNA amplified by PCR is not deteriorated. As the filter material, polyethylene, PTFE, etc. are suitable, and may be porous or hydrophobic. The dimensions of the first filter 228 and the second filter 230 are formed to fit into the filter installation space formed in the substrate 214 without any gaps.

[0075] In the substrate 214, a first branched channel 231 is formed, which branches off from the channel 212 at a first branching point 212c between the first filter 228 and the second filter 230. A first sample introduction port 233 is formed at the position of an end 231a of the first branched channel 231 in the substrate 214 (see FIG. 15). In addition, in the substrate 214, a second branched channel 232 is formed, which branches off from the channel 212 at a second branching point 212d between the first branching point 212c and the second filter 230. A second sample introduction port 234 is provided at the position of an end 232a of the second branched channel 232 in the substrate 214. The first sample introduction port 233 and the second sample introduction port 234 are formed so as to be exposed to an upper surface 214b of the substrate 214.

[0076] The portion of the flow channel 212 between the first filter 228 and the first branch point 212c forms a thermal cycle region 212e, which is scheduled to have a high temperature region and a medium temperature region, in order to thermally cycle the sample. The thermal cycle region 212e of the flow channel 212 includes a serpentine flow channel. This is to efficiently provide the heat provided by the PCR device in the PCR process to the sample, and to ensure that the volume of the sample that can be subjected to PCR is at least a certain amount. The thermal cycle region 212e includes a pair of reaction regions, each of which includes a serpentine flow channel, and a connection region that connects the pair of reaction regions.

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

[0078] In the PCR reaction vessel 210 according to the second embodiment, most of the flow channel 212 is formed in a groove shape exposed on the lower surface 214a of the substrate 214. This is to make it easy to mold by injection molding using a mold or the like. In order to utilize this groove as a flow channel, a flow channel sealing film 216 is attached to the lower surface 214a of the substrate 214. The flow channel sealing film 216 may have adhesiveness on one main surface, or a functional layer that exerts adhesiveness or adhesion by pressing may be formed on one main surface, and has a function of easily adhering to and integrating with the lower surface 214a of the substrate 214. It is desirable that the flow channel sealing film 216 is formed from a material having low autofluorescence, including the adhesive. In this respect, a transparent film made of a resin such as cycloolefin polymer, polyester, polypropylene, polyethylene, or acrylic is suitable, but is not limited to these. The flow 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] In the PCR reaction container 210 according to the second embodiment, the first air communication port 224, the second air communication port 226, the first sample introduction port 233, the second sample introduction port 234, the first filter 228, and the second filter 230 are exposed on the upper surface 214b of the substrate 214. Therefore, the first sealing film 218 is attached to the upper surface 214b of the substrate 214 to seal the first air communication port 224 and the first filter 228. In addition, the second sealing film 220 is attached to the upper surface 214b of the substrate 214 to seal the second air communication port 226 and the second filter 230. In addition, the third sealing film 222 is attached to the upper surface 214b of the substrate 14 to seal the first sample introduction port 233 and the second sample introduction port 234.

[0080] The first sealing film 218 has a size capable of simultaneously sealing the first air communication port 224 and the first filter 228, and the second sealing film 220 has a size capable of simultaneously sealing the second air communication port 226 and the second filter 230. A pressurized pump (described later) is connected to the first air communication port 224 and the second air communication port 226 by perforating the first air communication port 224 and the second air communication port 226 with a hollow needle (a syringe needle with a sharp tip) provided at the tip of the pump. For this reason, the first sealing film 218 and the second sealing film 220 are preferably made of a material and a thickness that allows easy perforation by a needle. In the second embodiment, a sealing film of a size capable of simultaneously sealing the corresponding air communication port and the filter is described, but a mode in which these are sealed separately is also acceptable. Also, a sealing film capable of sealing the first air communication port 224, the first filter 228, the second air communication port 226, and the second filter 230 together (as one piece) may be used.

[0081] The third sealing film 222 is used in a size that can simultaneously seal the first sample introduction port 233 and the second sample introduction port 234. The introduction of the sample into the flow channel 212 through the first sample introduction port 233 and the second sample introduction port 234 is performed by once peeling the third sealing film 222 from the substrate 214, and after the introduction of a predetermined amount of sample, the third sealing film 222 is returned and attached to the upper surface 214b of the substrate 214 again. For this reason, the third sealing film 222 is preferably a film having adhesiveness that can withstand several cycles of attachment / detachment. The third sealing film 222 may be a film in which a new film is attached after the introduction of the sample, in which case the importance of the characteristics related to attachment / detachment may be reduced. In addition, in the second embodiment, a sealing film of a size that can simultaneously seal the first sample introduction port 233 and the second sample introduction port 234 has been described, but these may be sealed separately.

[0082] The first sealing film 218, the second sealing film 220, and the third sealing film 222 may have an adhesive layer formed on one main surface, or a functional layer that exerts adhesiveness or adhesion by pressing, similar to the flow path sealing film 216. The first sealing film 218, the second sealing film 220, and the third sealing film 222 are preferably formed from a material having low autofluorescence, including the adhesive. In this respect, a transparent film made of a resin such as cycloolefin (COP), polyester, polypropylene, polyethylene, or acrylic is suitable, but is not limited to these. As described above, it is also desirable that the adhesiveness and other properties do not deteriorate to a degree that affects use even after multiple attachment / detachment, but in the case of an embodiment in which a new film is attached after peeling and introduction of a sample, the importance of the properties related to attachment / detachment may be mitigated.

[0083] Next, a method of using the PCR reaction vessel 210 configured as above will be described. First, a sample to be amplified by thermal cycling is prepared. The sample may be 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, the third sealing film 222 is peeled off from the substrate 214, and the first sample introduction port 233 and the second sample introduction port 234 are opened.

[0084] Next, a sample is introduced into either the first sample introduction port 233 or the second sample introduction port 234 using a dropper, syringe, or the like. Figure 18 shows a schematic diagram of a state in which a sample 270 has been introduced into a PCR reaction vessel 210. In Figure 18, in order to emphasize the position of the sample 270, the sample 270 is represented by a solid line thicker than that of the flow channel 212. It should be noted that this does not represent a state in which the sample 270 protrudes from the flow channel.

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

[0086] 19, the third sealing film 222 is attached back to the substrate 214 to seal the first sample introduction port 233 and the second sample introduction port 234. A new third sealing film 222 may be attached as described above. This completes the introduction of the sample 270 into the PCR reaction container 210.

[0087] Fig. 20 is a diagram illustrating a PCR device 300 using a PCR reaction container 210. Fig. 21 is a diagram illustrating a state in which the PCR reaction container 210 is set in a predetermined position in the PCR device 300.

[0088] The PCR device 300 includes a fluorescence detection optical probe 2122, a first heater 2134, and a second heater 2135. As shown in Fig. 21, the PCR reaction vessel 210 is placed in the PCR device 300 such that a pair of reaction regions in the thermal cycle region 212e of the flow channel 212 are disposed on the first heater 2134 and the second heater 2135, and the fluorescence detection optical probe 2122 is disposed in a connection region between the pair of reaction regions. The PCR device 300 can use the PCR device applied to the PCR reaction vessel according to the first embodiment.

[0089] The PCR device 300 further includes a pump system 2110 for reciprocating the sample 270 in the thermal cycle region 212e. The pump system 2110 includes 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 communication 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 communication port 226 of the PCR reaction vessel 210. A specific method of connecting the nozzle and the air communication port will be described later. The pump system 2110 controls the pressure in the flow channel 212 via the first air communication port 224 and the second air communication port 226 to move the sample in the thermal cycle region 212e.

[0090] In the PCR device 300 according to the second embodiment, the first heater 2134 and the second heater 2135 are set to different temperatures. Each heater provides heat to individually control the temperature of a pair of reaction regions in the thermal cycle region 212e, and may be a means or configuration such as resistance heating or a Peltier element. For example, the first heater 2134 is controlled by the first heater driver 2130 so as to maintain the temperature of the reaction region on the right side of the paper in the thermal cycle region 212e of the flow channel 212 at a constant 94°C. The second heater 2135 is controlled by the second heater driver 2132 so as to maintain the temperature of the reaction region on the left side of the paper 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. In this way, 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 controlling the temperature of the thermal cycle region 212e, and may include other elements that improve the controllability of the temperature. This temperature control unit can divide the thermal cycle region 212e of the flow path 212 into two regions with different ambient temperatures. A temperature sensor (not shown) such as a thermocouple that measures the temperature at the corresponding location may be included near each heater, and other components that improve the controllability of the temperature may be included. Hereinafter, the reaction region in the flow path 212 with an ambient temperature of 94°C is referred to as the "high temperature region 2111", and the reaction region in the flow path 212 with an ambient temperature of 60°C is referred to as the "medium temperature region 2112". In the present embodiment, a PCR device equipped with a PCR reaction vessel having a thermal cycle region in which two temperature levels are set as two reaction regions and a temperature control unit will be described in detail, but a PCR device equipped with a PCR reaction vessel having a thermal cycle region in which three or more temperature levels can be set and a temperature control unit may be used. In this case (not shown), as an example, a PCR device equipped with a PCR reaction vessel having reaction regions arranged from the left side of the paper as a low temperature section, a medium temperature section, and a high temperature section and a temperature control unit may be used. In such a case, for example, the low temperature section is controlled to be maintained at 50 to 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 arranged to reciprocate the sample 270 in the thermal cycle region 212e of the flow channel 212. The control unit 2107 alternately operates the first pump 2103 and the second pump 2104 under certain conditions through the first driver 2105 and the second driver 2106, so that the sample 270 can be reciprocated between the high temperature section 2111 and the medium temperature section 2112 of the flow channel 212, and the sample 270 can be subjected to a thermal cycle under certain conditions. In the PCR device 300 according to the second embodiment, the first pump 2103 and the second pump 2104 are air pumps or blower pumps of a type in which the air pressures on the primary side and the secondary side become equal instantly when either of them is stopped, and when either of them is stopped, the air pressures on the primary side and the secondary side become equal. If such a type of pump is not used, that is, if a pump that maintains the pressure immediately before the pump is stopped is used, a phenomenon occurs in which the sample continues to move slightly even when the pump is stopped, and the sample does not stop in the specified reaction region, and the temperature of the sample cannot be controlled appropriately. On the other hand, when the reactor is stopped (open), the outside air is air-pressure-equal to atmospheric pressure between the reactor and the flow path of the PCR reaction vessel; however, a filter is provided between the air communication port and the flow path, preventing contamination of the flow path.

[0092] PCR can be performed on the sample 270 by the above-mentioned thermal cycle, and the fluorescence from the sample 270 in the flow path can be detected, and the value can be used as an index for judging the progress of PCR and the end of the reaction. The optical probe 2122 for detecting fluorescence and the driver 2121 can be an optical fiber type fluorescence detector FLE-510 manufactured by Nippon Sheet Glass Co., Ltd., which has a very compact optical system, can perform rapid measurement, and can detect fluorescence regardless of the light or dark atmosphere. This optical fiber type fluorescence detector can be easily arranged in a narrow space between two temperature regions in the thermal cycle region. This optical fiber type fluorescence detector can be tuned to have the wavelength characteristics of its excitation light / fluorescence suitable for the fluorescent characteristics of the sample 270, and can provide an optimal optical / detection system for samples having various characteristics. In addition, the optical probe 2122 for detecting fluorescence and the driver 2121 may be provided at multiple locations across the thermal cycle region 212e. For example, they may be installed to detect fluorescence from the sample 270 in the flow path in the high temperature section 2111 or the medium temperature section 2112. In addition to the function of obtaining information for judging the progress and completion of PCR, it can also function as a position sensor for reliably detecting whether the sample 270 is in the high temperature section 2111 or the medium temperature section 2112 or not.

[0093] In the PCR device 300 configured as above, the control unit 2107 of the pump system 2110, the driver 2121 of the optical probe 2122 for detecting fluorescence, the first heater driver 2130, and the second heater driver 2132 are controlled by the CPU 2141 so as to operate optimally. When the reaction region in which three levels of temperature are set as described above is provided, a third heater driver (not shown) is also controlled by the CPU in addition to the above.

[0094] Fig. 22 is a diagram showing a state in which the nozzle of the pump system and the air communication port of the PCR reaction vessel are connected. Fig. 23 is a CC cross-sectional view of the PCR reaction vessel 210 shown in Fig. 23. As described above, the first nozzle 2101 is connected to the first air communication port 224, and the second nozzle 2102 is connected to the second air communication port 226.

[0095] 23, a needle 2150 is provided at the tip of the first nozzle 2101. The first nozzle 2101 is connected to the first air communication port 224 by piercing the first sealing film 218 with this needle 2150. The second nozzle 2102 and the second air communication port 226 are connected in a similar manner.

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

[0097] FIG. 24 shows a state in which 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 path 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 FIG. 24, the second pump 2104 connected to the second nozzle 2102 is operated, and the first pump 2103 connected to the first nozzle 2101 is stopped. That is, the first air communication port 224 connected to the first nozzle 2101 extending from the first pump 2103 is open to atmospheric pressure. When the second pump 2104 is operated to send air from the second nozzle 2102 to the second air communication port 226, the sample 270 moves from the buffer flow path region 212f of the flow path 212, passes through the medium temperature section 2112, and moves to the high temperature section 2111. This state is defined as the initial state.

[0098] More specifically, at the same time as or immediately before the start of the operation of the second pump 2104, the fluorescence emitted from the flow path is monitored using the optical probe for detecting fluorescence 2122. When there is nothing at the measurement point of the optical probe for detecting fluorescence 2122, the detected fluorescence is zero or at the background level, but when the sample 270 is present at the measurement point, the fluorescence is detected. Therefore, the monitoring of the fluorescence is started from the start of the 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 moving to the high temperature section 2111, and the operation of the second pump 2104 is stopped at this point, thereby completing the setting of the initial state. Also, if the optical probe for detecting fluorescence 2122 is located further in the high temperature section 2111, the sample 270 can be more reliably stopped at 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 according to the amount of sample to be subjected to PCR processing, a desired constant amount of sample can always be sent to the thermal cycle region 212e of the channel 212, and the amount of fluorescence that affects the progress and termination judgment of PCR can be made approximately constant. That is, 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 the initial state, the sample 270 is subjected to a thermal cycle to allow PCR to proceed. Measurement of fluorescence by the fluorescence detection optical probe 2122 continues.

[0101] (A) First, the sample 270 is placed in the high temperature section 2111 (atmosphere of about 94° C.) for 1 to 30 seconds (Deneturation: thermal denaturation step). This step denatures double-stranded DNA into single strands.

[0102] (B) Next, the first pump 2103 connected to the first nozzle 2101 is operated to move the sample 270 to the medium temperature section 2112 (atmosphere of about 60° C.). Specifically, the sample 270 is pushed from the high temperature section 2111 toward the medium temperature section 2112 by the action of the first pump 2103. Since the fluorescence measurement by the optical probe for detecting fluorescence 2122 continues, the operation of the first pump 2103 is stopped when the amount of fluorescence rises from the background level as the sample 270 passes through the measurement point of the optical probe for detecting fluorescence 2122 and then falls again (or when a certain time has passed since the amount of fluorescence fell). Also, when the optical probe for detecting fluorescence 2122 is in the medium temperature section 2112, the sample 270 can be more reliably stopped in the medium temperature section 2112.

[0103] (C) In the medium temperature section 2112, the sample 270 is kept waiting for 3 to 60 seconds (Annealing+Elongation: Annealing step+Elongation step). Through this step, the primers previously contained in the sample 270 are bound to the sample 270, resulting in further elongated DNA.

[0104] (D) Next, the second pump 2104 connected to the second nozzle 2102 is operated to move the sample 270 from the medium temperature section 2112 to the high temperature section 2111. The timing to stop the pump operation is determined from the fluctuation in the amount of fluorescence measured by the fluorescence detection optical probe 2122, as described above. After the sample 270 is moved to the high temperature section 2111, it is left to stand for 1 to 30 seconds to be thermally denatured.

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

[0106] After the predetermined number of thermal cycles are completed, the first pump 2103 and the second pump 2104 are stopped to terminate the PCR. Even when the predetermined number of thermal cycles are being applied, the 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 the concentration of the sample 270 to be accurately known.

[0107] According to the PCR reaction vessel 210 of the second embodiment, the first filter 228 is provided between the first air communication port 224 and the flow path 212, and the second filter 230 is provided between the second air communication port 226 and the flow path 212, thereby preventing contamination in the flow path 212. Although taking measures to prevent contamination on the pump system 2110 side tends to be costly, the PCR reaction vessel 210 of the second embodiment can prevent contamination only on the PCR reaction vessel 210 side, which is economical. Furthermore, when the PCR reaction vessel is used as a disposable vessel, the filter is always new, so that contamination can be further prevented at a low cost. Furthermore, regarding disposal of the PCR reaction vessel, since the sample is substantially sealed in the PCR reaction vessel, it is also meaningful in terms of safety and the environment.

[0108] Furthermore, according to the PCR reaction vessel 210 of the second embodiment, by providing a buffer flow path region in the flow path 212, the sample to be subjected to PCR can be dispensed and only the required amount of sample can be sent to the thermal cycle region of the flow path 212 at any one time.

[0109] In the PCR device 300 according to the second embodiment, the first pump 2103 and the second pump 2104, whose primary and secondary pressures are equal when stopped, are alternately operated to move the sample back and forth within the flow path 212 of the PCR reaction vessel 210. In this case, since excessive pressure is not applied to the sample during liquid transfer (pressure is applied to the sample in the flow path), and pressure within the flow path is not reduced, evaporation and boiling (foaming) of the liquid containing the sample due to the action of the high temperature part 2111 can be prevented.

[0110] Furthermore, in the PCR device 300 according to the second embodiment, the fluorescence from the sample is constantly monitored in the thermal cycle region even during PCR (real-time PCR). This allows the end timing of PCR to be determined based on the measured amount of fluorescence. Furthermore, the passage of the sample can be detected by monitoring the change in fluorescence using the fluorescence detection optical probe 2122, and the alternating operations of the first pump 2103 and the second pump 2104 can be controlled based on the change in the amount of fluorescence 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 cycle region.

[0111] On the other hand, in the case of a PCR reaction vessel and a PCR device having a reaction region with three temperature levels controlled, namely, a high temperature region, a medium temperature region, and a low temperature region, it becomes possible to carry out each step of thermal denaturation in the high temperature region, annealing in the medium temperature region, and extension in the low temperature region, and the control of these steps can be easily developed and improved by a person skilled in the art based on the above detailed explanation. In addition, whether the reaction region has two or three levels can be appropriately selected by a person skilled in the art depending on the characteristics of the sample.

[0112] The present invention has been described above based on the embodiment. This embodiment is merely an example, and it will be understood by those skilled in the art that various modifications are possible in the combination of each component and each treatment process, and that such modifications are also within the scope of the present invention.

[0113] In the above embodiment, a pair of pumps are arranged at both ends of the flow path, so that the pressure on the primary side and the secondary side are equal when the pumps are stopped. However, a pump capable of pressurizing and suctioning may be provided at only one end of the flow path, and the other end may be open to atmospheric pressure. That is, the sample is moved within the thermal cycle region by controlling the pressure within the flow path via the first air communication port or the second air communication port. In this case, the process of switching the operation of the pair of pumps at a fixed timing is not required, making it easier to control the pumps.

[0114] In the above embodiment, the measurement point of the optical probe for detecting fluorescence is disposed between the high temperature part and the medium temperature part, but the measurement points of the optical probe for detecting fluorescence may be disposed in both the high temperature part and the medium temperature part, in which case the positioning accuracy of the sample can be improved. [Explanation of symbols]

[0115] 10, 210 PCR reaction vessel, 12, 212 flow path, 14, 214 substrate, 16, 216 flow path sealing film, 18, 218 first sealing film, 20, 220 second sealing film, 22, 222 third sealing film, 24, 224 first air communication port, 26, 226 second air communication port, 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 flow path, 132, 2132 second heater driver, 133 sample introduction port, 134, 2134 first heater, 135, 2135 second heater, 141, 2141 CPU, 231 first branch flow path, 232 second branch flow path, 233 first sample introduction port, 234 second sample introduction port. [Industrial Applicability]

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

Claims

1. A resin substrate; A flow path formed in the resin substrate; A pair of filters provided at both ends of the flow path; A pair of air communication ports communicating with the flow path through the filter; a sample inlet for introducing a sample containing DNA into the flow channel; a thermal cycle region formed between the pair of filters in the flow path, the thermal cycle region including a plurality of different temperature regions capable of inducing PCR on a sample, and a connection region connecting the plurality of temperature regions; A PCR reaction vessel comprising: A temperature control unit for controlling the temperature of the thermal cycle area; a pump system connected to the air communication port and configured to control a pressure in the flow channel in order to move a sample in the thermal cycle region; at least one fluorescence detector that detects fluorescence from a sample in the thermal cycling region; Equipped with the fluorescence detector includes fluorescence detection optics; the fluorescence detection optical device is disposed in a direction normal to a surface of the resin substrate from a flow path belonging to the thermal cycle region and spaced apart from the surface of the resin substrate; The PCR device is characterized in that the fluorescence detector includes a fluorescence detector arranged to detect fluorescence from a sample within one of the connection areas through which the sample passes when moving from one of the temperature areas to another adjacent one of the temperature areas.

2. The pump system includes a pair of pumps; 2. The PCR device according to claim 1, wherein the pair of pumps are connected to the pair of air communication ports, respectively, and the sample is moved back and forth through the plurality of temperature zones by alternately operating either one of the pumps.

3. 3. The PCR device according to claim 1, wherein the plurality of temperature regions include a high-temperature section capable of thermally denaturing DNA contained in a sample, and a medium-temperature section capable of annealing and elongating the DNA.

4. 4. The PCR device according to claim 3, wherein the fluorescence detector comprises a fluorescence detector arranged to detect fluorescence from a sample in either the high temperature section or the intermediate temperature section.

5. 5. The PCR apparatus according to claim 1, wherein the pump system is controlled based on a value detected by any one of the fluorescence detectors.

6. The filter is disposed within the resin substrate, 6. The PCR device according to claim 1, wherein the filter prevents contamination caused by the pump system.

7. The PCR reaction vessel is detachable from the PCR device; The thickness of the filter is smaller than the thickness of the resin substrate constituting the PCR reaction vessel, 7. The PCR device according to claim 1, wherein the filter is accommodated in a filter installation space provided in the resin substrate.

8. 8. The PCR device according to claim 1, wherein the PCR reaction vessel is provided with a sealing film for sealing the air communication port.

9. the pump system includes a hollow needle in connection with the air communication port; 9. The PCR device according to claim 8, wherein the pump system and the flow path are communicated with each other by piercing the sealing film with the needle.

10. preparing a PCR reaction vessel including a resin substrate, a flow path formed in the resin substrate, a pair of filters provided at both ends of the flow path, a pair of air communication ports communicating with the flow path through the filters, a sample introduction port for introducing a sample containing DNA into the flow path, and a thermal cycle region formed between the pair of filters in the flow path, the thermal cycle region including a plurality of different temperature regions capable of causing PCR in the sample, and a connection region connecting the plurality of temperature regions; introducing a sample into the PCR reaction vessel through the sample introduction port; a step of setting the PCR reaction vessel in a PCR device including a temperature control unit for controlling the temperature of the thermal cycle region in the PCR reaction vessel, a pump system including a pair of pumps, the pair of pumps being connected to the pair of air communication ports, respectively, to control the pressure in the flow path in order to move the sample in the thermal cycle region, and at least one fluorescence detector for detecting fluorescence from the sample in the thermal cycle region; moving the sample back and forth between the plurality of temperature zones by alternately operating either one of the pair of pumps; Equipped with the fluorescence detector includes fluorescence detection optics; the fluorescence detection optical device is disposed in a direction normal to a surface of the resin substrate from a flow path belonging to the thermal cycle region and spaced apart from the surface of the resin substrate; A PCR method characterized in that the fluorescence detector includes a fluorescence detector arranged to detect fluorescence from a sample within one of the connection areas through which the sample passes when moving from one of the temperature areas to another adjacent one of the temperature areas.

11. 11. The PCR method of claim 10, wherein the fluorescence detector comprises a fluorescence detector positioned to detect fluorescence from a sample in at least one temperature region of the thermal cycling region.

12. The PCR method according to claim 10 or 11, characterized in that the multiple temperature regions include a high temperature section capable of thermally denaturing DNA contained in a sample, and a mid-temperature section capable of annealing and elongation.

13. 13. The PCR method of claim 12, wherein the fluorescence detector comprises a fluorescence detector positioned to detect fluorescence from a sample in either the high temperature section or the intermediate temperature section.

14. the PCR reaction vessel includes an air communication port sealing film for sealing the air communication port, the pump system includes a hollow needle in connection with the air communication port; 14. The PCR method according to claim 10, wherein the pump system and the flow channel are connected to each other by piercing the air communication port sealing film with the needle.

15. the PCR reaction vessel includes an air communication port sealing film for sealing the air communication port, and a sample introduction port sealing film for sealing the sample introduction port, The step of introducing the sample includes: peeling off the sample introduction port sealing film to open the sample introduction port; 15. The PCR method according to claim 10, further comprising a step of peeling off the air communication port sealing film to open at least one of the air communication ports.

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