PCR device

Abstract

To provide a PCR device of simple structure that allows heating, cooling, and measurement to be performed at one site.SOLUTION: The PCR device comprises: a holding part 2 capable of holding a sheet-like reaction container 1 having a chamber 10 which accommodates a sample solution; a temperature control part 5X capable of adjusting the temperature of the sample solution in the chamber 10 of the reaction container 1 held in the holding part 2 to a plurality of temperatures; and an optical measurement part 6X capable of measuring fluorescence characteristics of the sample solution in the chamber 10. At least a part of the temperature adjustment range of the sample solution in the chamber 10 by the temperature control part 5X overlaps with at least a part of the measurement range of the sample solution in the chamber 10 by the optical measurement part 6X.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present disclosure relates to a PCR device for analyzing nucleic acids contained in a test solution. [Background technology]

[0002] PCR (polymerase chain reaction) has been known as a method for amplifying nucleic acids (DNA) by mixing necessary reagents with template DNA to amplify a large amount of specific DNA in a short period of time, and various PCR devices using this method have been developed and commercialized. In recent years, improvements have been made to this PCR method, and real-time PCR methods and devices have been proposed that can measure the amount of amplified DNA in real time, for example, by fluorescence analysis (see, for example, Patent Document 1).

[0003] Demand for PCR genetic testing has increased significantly in recent years. During the COVID-19 pandemic in 2021, PCR testing centers were established throughout urban areas, resulting in a total of 55.39 million PCR tests in 2021, and the market size for genetic testing reaching 144.8 billion yen in 2021.

[0004] For such genetic testing as a disease prevention measure, rapid results are desirable, and therefore a system that is easy for testers to use is desirable. Large, high-precision PCR devices for research use are limited in the locations where they can be installed, and the personnel capable of operating such devices are also limited, making them unsuitable for rapid testing that cannot be performed anywhere (POCT: Point of Care Test). In other words, what is required for clinical PCR devices is a system that can be easily installed in any facility (even a small hospital), can be operated by doctors, nurses, or office staff, and is affordable and allows for easy testing.

[0005] A compact and simple PCR device for such clinical testing has been developed (see Non-Patent Document 1). The PCR device described in Non-Patent Document 1 uses a microchannel, and as the sample flows through the channel, it moves back and forth between a heating section and a cooling section, achieving the thermal cycle (repeated temperatures of 95 to 60°C) essential for PCR reactions. This type of thermal cycle appears simple, as the heating section only needs to heat and the cooling section only needs to cool. However, it requires highly accurate pump control to accurately move the pump back and forth through the highly accurate channel. In addition, excitation light must be irradiated onto the microchannel and the reflected light must be received by a sensor with high precision, which inevitably leads to a complex optical system. Therefore, although the PCR device described in Non-Patent Document 1 has been significantly miniaturized, there are limitations, and it has not yet achieved a cost-effectiveness that would allow for large-volume testing. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2021-159011 [Non-patent literature]

[0007] [Non-Patent Document 1] Gofoton Co., Ltd., product information, introduction website for the real-time PCR device "PicoGene (registered trademark) PCR1100", https: / / pcr.gofoton.co.jp / pcr1100-feature.html Summary of the Invention [Problem to be solved by the invention]

[0008] In light of this situation, the inventors have conducted extensive research into technology that allows for heating, cooling, and measurement in one location without the need to move the sample with high precision, and as a result, have completed the present invention, which relates to a PCR device with a simple configuration that allows for heating, cooling, and measurement in one location.

[0009] The present disclosure provides a PCR device with a simple configuration that allows heating, cooling, and measurement in one place. [Means for solving the problem]

[0010] The PCR device of the present disclosure comprises a holding section capable of holding a sheet-shaped reaction vessel having a chamber in which a test liquid is contained, a temperature adjustment section capable of adjusting the temperature of the test liquid in the chamber of the reaction vessel held in the holding section to a plurality of temperatures, and an optical measurement section capable of measuring the fluorescent characteristics of the test liquid in the chamber, wherein at least a portion of the temperature adjustment range of the test liquid in the chamber by the temperature adjustment section overlaps with at least a portion of the measurement range of the test liquid in the chamber by the optical measurement section. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a front view of the PCR device of the first embodiment placed on a table or the like, as viewed from above. [Figure 2] Fig. 2A is a plan view showing a sheet-like reaction vessel 1 for containing a test solution, and Fig. 2B is a cross-sectional view taken along the line II-II in Fig. 2A. [Figure 3] 2 is a front view showing the holder 2, the thermal unit 5, and the optical measurement unit 6 housed inside the housing 3. FIG. [Figure 4] FIG. 2 is a perspective view showing a thermal unit 5 and an optical measurement unit 6 separated from each other. [Figure 5] 10 is a cross-sectional view taken along the XY direction passing through the middle of the thermal unit 5 and the optical measurement unit 6 in the Z direction. [Figure 6] FIG. 6 is a cross-sectional view corresponding to FIG. 5 according to a modified example. [Figure 7] FIG. 5 is a cross-sectional view corresponding to FIG. 4 according to a modified example. [Figure 8] FIG. 10 is an explanatory diagram showing an example of overshoot control during temperature rise according to a modified example. [Figure 9] FIG. 10 is an explanatory diagram showing an example of overshoot control during temperature decrease according to a modified example. DETAILED DESCRIPTION OF THE INVENTION

[0012] [First embodiment] The PCR device according to the first embodiment of the present disclosure will be described below. In the drawings, the X, Y, and Z directions may be illustrated for ease of understanding.

[0013] <Structure of reaction vessel 1> FIG. 2A is a plan view showing a sheet-like reaction vessel 1 containing a test solution. FIG. 2B is a cross-sectional view taken along the line II-II in FIG. 2A. As shown in FIGS. 2A and 2B, the reaction vessel 1 used in the PCR device of the first embodiment is sheet-like, and its thickness dimension D1 is smaller than its width dimension D2 and depth dimension D3. In the first embodiment, D1 is approximately 0.7 mm, D2 is approximately 25.0 mm, and D3 is approximately 46.0 mm, but these dimensions are not limited thereto and can be modified as appropriate. The sheet-like reaction vessel 1 is rectangular in plan view when viewed parallel to the thickness direction (Y), but the shape can be modified. The reaction vessel 1 has a chamber 10, which is a void portion that contains a test solution containing a sample. In the first embodiment, the chamber 10 is cylindrical and appears circular in plan view, but this is not limited thereto. The shape of the chamber 10 can be modified as long as its thickness dimension is smaller than its width and depth dimensions. In the first embodiment, the chamber 10 has a cylindrical shape with a diameter of 8 mm, but this can be changed as appropriate. The chamber 10 can also be said to be a reaction chamber that holds test solutions that are adjusted to a plurality of temperatures.

[0014] As shown in FIG. 2B, the reaction vessel 1 has a bendable flexibility and is formed by a plurality of members. The reaction vessel 1 of the first embodiment includes a spacer 11 that forms the peripheral wall of the chamber 10, a first member 12 that is attached to the first side Y1 in the thickness direction (Y) of the spacer 11 and forms the chamber wall surface of the first side Y1 that forms the chamber 10, and a second member 13 that is attached to the second side Y2 in the thickness direction (Y) of the spacer 11 and forms the chamber wall surface of the second side Y2 that forms the chamber 10. As the spacer 11, a silicon tape having a hole that becomes the chamber 10 can be used, and as the first member 12 and the second member 13, a PCR seal can be used. When using the reaction vessel 1, it may be possible to attach the second member 13 to the spacer 11, fill the chamber 10 with a test solution containing a sample, and attach the first member 12 to the spacer 11 to seal the chamber 10.

[0015] <Structure of PCR Device> FIG. 1 is a front view of the PCR device placed on a table in the first embodiment as viewed from above. As shown in FIG. 1, the PCR device has a holding portion 2 capable of holding the reaction vessel 1. The holding portion 2 has a slit 20 into which the sheet-like reaction vessel 1 can be inserted. The slit 20 has a rectangular parallelepiped portion corresponding to the outer shape of the reaction vessel 1, and guides the reaction vessel 1 so that it can be in a flat plate shape. The opening of the slit 20 is open to the outside of the housing 3 of the PCR device, and the reaction vessel 1 is configured to be detachable (insertable and removable) from the outside of the housing 到 3 into the slit 20. The PCR device has a touch panel 4 that also serves as a display and an operation unit, and a processor (not shown) that controls the equipment including the touch panel 4.

[0016] FIG. 3 is a front view showing the holder 2, thermal unit 5, and optical measurement unit 6 housed inside the housing 3. In FIG. 3, the reaction vessel 1 inserted into the slit 20 is shown hatched. FIG. 4 is a perspective view showing the thermal unit 5 and optical measurement unit 6 separated from each other. FIG. 5 is a cross-sectional view taken along the XY direction passing through the middle of the thermal unit 5 and optical measurement unit 6 in the Z direction. As shown in FIGS. 3 and 4, the PCR device has a thermal unit 5 capable of adjusting the temperature of the test liquid in the chamber 10 of the reaction vessel 1 held in the holder 2, and an optical measurement unit 6 capable of measuring the fluorescence properties of the test liquid in the chamber 10.

[0017] As shown in FIGS. 3 to 5, the thermal unit 5 has a thermal case 50, and the optical measurement unit 6 has a measurement case 60. The thermal case 50 and the measurement case 60 are fixed together with fasteners such as screws. A recess that becomes the slit 20 is formed between the thermal case 50 and the measurement case 60. By combining the thermal case 50 and the measurement case 60, the measurement case 60 forms a wall surface 21 on the first side Y1 of the slit 20, and the thermal case 50 forms a part (22) of the wall surface on the second side Y2 of the slit 20. The optical measurement unit 6 is disposed on the first side Y1 in the thickness direction (Y) of the reaction vessel 1 inserted into the slit 20, and the thermal unit 5 is disposed on the second side Y2 in the thickness direction (Y) of the reaction vessel 1.

[0018] <Thermal Unit 5> The thermal unit 5 has a heat transfer surface 51 that can come into contact with the reaction vessel 1. The heat transfer surface 51 is arranged as a part of the wall surface on the second side Y2 that forms the slit 20 in the thermal case 50. The heat transfer surface 51 is a surface on the first side Y1 of a heat transfer member 52, such as a copper plate, that has a higher thermal conductivity than the thermal case 50, and is a flat surface in the first embodiment. The heat transfer surface 51 protrudes toward the first side Y1 beyond the wall surface 22 surrounding the heat transfer surface 51 among the wall surfaces that form the slit 20 in the thermal case 50, making it easier to come into contact with the reaction vessel 1. The heat transfer member 52 having the heat transfer surface 51 is a rectangular plate measuring 20 mm × 20 mm × 1 mm, and the heat transfer surface 51 is larger than the bottom surface (surface on the second side Y2) of the chamber 10, but is not limited thereto. It is sufficient that the area of ​​the heat transfer surface 51 is equal to or larger than the area of ​​the chamber 10 (area on the second side Y2). As a result, the temperature of the reagent solution in the chamber 10 can be adjusted over the entire chamber 10 as a reaction chamber.

[0019] 5, the thermal unit 5 has a Peltier element 53 capable of heating and cooling. The Peltier element 53 is thermally connected to the surface of the second side Y2 of the heat transfer member 52. When a current flows through the Peltier element 53 in a first direction, the surface of the Peltier element 53 on the first side Y1 generates heat, and the surface of the Peltier element 53 on the second side Y2 is cooled. On the other hand, when a current flows through the Peltier element 53 in a second direction opposite to the first direction, the surface of the Peltier element 53 on the first side Y1 is cooled, and the surface of the Peltier element 53 on the second side Y2 generates heat.

[0020] 3 to 5, the thermal unit 5 includes a heat sink 54 thermally connected to the surface of the second side Y2 of the Peltier element 53, a fan 55 for dissipating heat from the heat sink 54 to the outside of the housing 3, and a temperature detection unit 56 such as a thermistor for detecting the temperature of the heat transfer member 52 including the heat transfer surface 51. The fan 55 faces the outside of the housing 3 through an exhaust port (not shown) of the housing 3, and is capable of dissipating heat from the heat sink 54 through the exhaust port. The processor is capable of controlling the current to the Peltier element 53, detecting temperature with the temperature detection unit 56, controlling the fan 55, and the like.

[0021] 5, the heat transfer surface 51 of the heat transfer member 52 is larger than the entire wall surface of the second side Y2 of the chamber 10, so that by controlling the temperature of the heat transfer member 52, it is possible to adjust the temperature of all the test liquids in the single chamber 10. The heat transfer member 52, the Peltier element 53, and the temperature detection unit 56 constitute a temperature adjustment unit 5X that can adjust the temperature of the test liquids. The temperature adjustment range of the test liquids in the chamber 10 by the temperature adjustment unit 5X is the entire chamber 10.

[0022] <Optical measurement unit 6> As shown in FIGS. 3 to 5, the optical measurement unit 6 has an optical measurement section 6X that can measure the fluorescence characteristics of the test liquid in the chamber 10. The optical measurement section 6X includes a light source 61 such as an LED that irradiates light (excitation light, etc.) toward the chamber 10, and an optical sensor 62 that detects light (excited fluorescence, etc.) emitted from the test liquid in the chamber 10. The optical measurement section 6X may also include an optical filter such as a bandpass filter. In the optical measurement section 6X of the first embodiment, the axis of the light irradiated from the light source 61 is different from the axis of the light reaching the optical sensor 62. The processor described above can control the optical measurement unit 6. The measurement range of the test liquid by the optical measurement section 6X is the entire area of ​​the chamber 10 serving as a reaction chamber, or a portion of the center thereof.

[0023] The measurement case 60 has a hollow storage space SP1 inside. The measurement case 60 houses the optical measurement unit 6X in the storage space SP1. The wall surface 21 of the measurement case 60 on the first side Y1 of the slit 20 has a light conducting port 63 that passes light from the chamber 10 to the optical measurement unit 6X. The light conducting port 63 is connected to the storage space SP1. The storage space SP1 is optically shielded from the outside to prevent noise light from entering from outside the measurement case 60, and is an enclosed space that is not connected to the outside except for the light conducting port 63 to prevent the air temperature in the storage space SP1 from cooling. As a result, the intrusion of external light can be suppressed or prevented, and deterioration of the measurement accuracy of the optical measurement unit 6X can be avoided. Further, as the temperature of the test solution is adjusted by the temperature adjustment unit 5X, the air in the accommodation space SP1 of the measurement case 60 is heated. Since the accommodation space SP1 has a sealed structure, it becomes a heat retention structure in which the heated air is difficult to escape, and the time to reach the target temperature in the temperature control by the temperature adjustment unit 5X can be shortened.

[0024] <Operation of the PCR device> The temperature adjustment unit 5X can adjust the temperature of the test solution in the chamber 10 of the reaction vessel 1 to a plurality of temperatures in order to perform PCR. Specifically, the temperature adjustment unit 5X is configured to be capable of performing temperature control in any one of a plurality of thermal modes including a first thermal mode for controlling the temperature of the heat transfer surface 51 to a first temperature and a second thermal mode for controlling the temperature of the heat transfer surface 51 to a second temperature. The first to second thermal modes form a set, and the temperature control of one set can be repeatedly executed. As an example, the first temperature may be 90°C to 105°C, and the second temperature may be 50°C to 70°C. The temperature range of 90°C to 105°C indicates the denaturation state, and the temperature range of 50°C to 70°C indicates the annealing and extension states. If an initial denaturation temperature is required, the first temperature may be provided once before the above set as necessary. The processor performs PID control on the current of the Peltier element 53 based on the temperature detected by the temperature detection unit 56 and the target temperature. In PID control, a current corresponding to the difference between the temperature detected by the temperature detection unit 56 and the target temperature of the heat transfer surface 51 is passed through the Peltier element 53. In the first embodiment, PWM (Pulse Width Modulation) control is used, and the processor changes the duty ratio according to the difference between the temperature detected by the temperature detection unit 56 and the target temperature of the heat transfer surface 51.

[0025] For example, when changing the target temperature of the heat transfer surface 51 from the first target temperature to a second target temperature higher than the first target temperature while controlling the target temperature of the heat transfer surface 51 to the first target temperature, it is possible to execute control to pass a current through the Peltier element 53 so that the surface of the first side Y1 of the Peltier element 53 generates heat and drive the fan 55. For example, when the target temperature of the heat transfer surface 51 is controlled to a first target temperature, and the target temperature of the heat transfer surface 51 is changed to a second target temperature lower than the first target temperature, a control can be executed to pass a current through the Peltier element 53 to cool the surface of the first side Y1 of the Peltier element 53 and to drive the fan 55.

[0026] <Modification> (A) The dimensions of the sheet-like reaction vessel 1 of the first embodiment may be changed as desired. For example, the thickness dimension D1 of the reaction vessel 1 is preferably set within a range of 0.5 mm or more and 1.4 mm or less. The width dimension D2 of the reaction vessel 1 is preferably set within a range of 8.0 mm or more and 25.0 mm or less. The depth dimension D3 of the reaction vessel 1 is preferably set within a range of 8.0 mm or more and 50.0 mm or less. The diameter of the circle in the cylindrical chamber 10 is preferably within a range of 4 mm to 12 mm. The shape of the chamber 10 is not limited to a cylindrical shape and can be changed as appropriate. The thickness of the chamber 10 may be, for example, 0.001 to 10 mm, or 0.01 to 3 mm. The area of ​​the chamber 10 is, for example, 50 mm. 2 It may be 900 mm2 or more, or 900 mm2 or more.

[0027] A test example regarding the circular diameter of the chamber 10 described above in (A) is as follows. the purpose The purpose of this test example is to examine the optimum diameter size of the reagent sealing portion of the chamber 10 and to confirm compatibility with the optical measurement portion 6X that detects fluorescence. method Samples were prepared using the following reagents: TaqMan Fast Advanced Master Mix, GAPDH primers / probes, and HEK293 cell-derived cDNA (adjusted to approximately 100 ng / μL using DEPC-treated water). Chambers 10 were fabricated from silicone tape to the predetermined size described in the first embodiment, and the reagents were sealed in with a PCR seal. PCR was then performed on each chamber using the PCR instrument of the first embodiment, and the results were compared. Analysis method To determine the enhancement value of the fluorescence intensity, the pulse count value was output, and the value at 40 cycles was divided by the value at 1 cycle to obtain the enhancement value ratio. The average value, standard deviation, and coefficient of variation of the fluorescence enhancement value were calculated, and the optimal diameter size of the chamber 10 was determined. The results are shown in Table 1 below. The average fluorescence enhancement ratio was low at 170% for a 6mm diameter chamber, but the averages for other sizes were 217-253%, with no significant difference. On the other hand, the chamber with an 8mm diameter had the lowest coefficient of variation for the fluorescence enhancement ratio, and the most stable fluorescence detection was possible, with a coefficient of variation of 8%. [Table 1] According to Table 1 above, the average value of fluorescence enhancement does not differ significantly between chamber 10 diameters of 4 to 12 mm, and it can be seen that this does not affect fluorescence detection. From the coefficient of variation values ​​for each chamber, it can be seen that a diameter of 8 mm enables more stable fluorescence detection.

[0028] (B) In the first embodiment, the chamber 10 is a single, sealed chamber, but is not limited to this. For example, an inlet for introducing a test solution into the chamber 10 of the reaction chamber may be provided, and the entrance of the inlet may be positioned outside the temperature adjustment range of the temperature adjustment unit 5X. For example, an outlet may be provided to remove air bubbles from the reaction chamber 10. When an inlet and an outlet are provided, the chamber can be sealed by closing the inlet and outlet with a sealant such as a seal after introducing the test solution.

[0029] (C) In the first embodiment, the holder 2 is a slit 20, but is not limited to this. For example, the housing 3 of the device may be provided with a slide holder that is movable between the inside and outside of the housing, and the slide holder may house the reaction vessel 1. The slide holder provides a light-blocking effect, which can improve the measurement accuracy of the optical measurement unit 6X. Furthermore, when the holding portion 2 is a slit 20, a lid may be provided that can close the opening of the slit 20. The lid may be a sliding type, a double-hinged type, or a single-hinged type.

[0030] (D) In ​​the first embodiment, the heating / cooling element (Peltier element 53) constituting the temperature adjustment unit 5X is disposed only on the second side Y2 of the reaction vessel 1, but this is not limiting. For example, a heating element may be provided not only on the second side Y2 of the reaction vessel 1 but also on the first side Y1. In this case, the heating element on the first side Y1 only needs to be transparent so that light from the optical measurement unit 6X can be transmitted therethrough. One example is a transparent film heater.

[0031] (E) In the first embodiment, the heat transfer surface 51 is a flat surface, but is not limited to this. For example, the center of the heat transfer surface 51 may be shaped to protrude more than the ends in order to bring the reaction vessel 1 into close contact with the heat transfer surface 51. Furthermore, the heat transfer surface 51 may be shaped so that its thickness increases as it advances in the depth direction of the slit 20.

[0032] (F) When the reaction vessel 1 is heated, the internal pressure of the chamber 10 increases, causing the reaction vessel 1 to expand in the thickness direction (Y). If the thickness of the slit 20 is made smaller than the thickness of the reaction vessel 1, the wall surface of the slit 20 can suppress the expansion of the chamber 10. However, resistance occurs when inserting the sheet-like reaction vessel 1 into the slit 20, making it difficult to properly insert the sheet-like reaction vessel 1 into the slit 20. Therefore, for example, a mechanical structure may be provided in which, after the reaction vessel 1 is inserted into the slit 20, a separately provided operating unit is operated to narrow the thickness of the slit 20. In this case, for example, the thermal unit 5 and the optical measurement unit 6 may be made movable in a direction in which they approach each other. As another idea, a pressing part may be provided that presses the reaction vessel 1 when the operating part is operated.

[0033] (G) FIG. 6 is a cross-sectional view corresponding to FIG. 5 according to a modified example. As shown in FIG. 6, when the reaction vessel 1 is held by the holder 2, a transparent plate 7 may be disposed between the light guide port 63 and the reaction vessel 1, and the transparent plate 7 may be harder than the reaction vessel 1. The hardness can be measured using a Shore A hardness tester. Heating the test solution in the chamber 10 increases the internal pressure of the chamber 10, and because the first side Y1 of the chamber 10 is open through the light guide port 63, the first member 12 tends to expand. The expansion of the reaction vessel 1 makes it more likely for bubbles to form. Therefore, by disposing the transparent plate 7, which is harder than the reaction vessel 1, on the first side Y1 of the first member 12, the reaction vessel 1 is prevented from expanding toward the first side Y1, and the reaction vessel 1 is more likely to come into close contact with the heat transfer surface 51 due to the internal pressure. This suppresses the expansion of the chamber 10, thereby suppressing the generation of bubbles, and improves heat transfer efficiency. For example, glass can be used for the transparent plate 7. The surface dimension of the transparent plate 7 is preferably larger than both the chamber 10 and the light guide port 63. The transparent plate 7 may be fixed to the device (optical measurement unit 6). The transparent plate 7 and the reaction vessel 1 may be fixed. The transparent plate 7 and the reaction vessel 1 may not be fixed, but may be inserted into the slit 20 at the same time.

[0034] A test example regarding the transparent plate 7 described above in (G) is as follows. the purpose In this test example, in order to solve the problem of improving fluorescence accuracy by suppressing bubble formation in the qPCR reagent, a transparent plate 7 as a slide glass was placed on the detection side of the reaction vessel 1, and the effect was evaluated. method In this test example, the THUNDERBIRD Probe One-step qRT-PCR Kit and β-Actin primer / probe were used. The template was total RNA extracted from human keratinocytes, adjusted to 10 ng / uL with DEPC-treated water. A chamber containing the relevant reagents was created using silicone tape and a PCR seal. To compare the effects of the presence or absence of a glass slide on bubble formation and fluorescence detection ability, PCR was performed with and without a glass slide inserted, and the results were compared. result The Ct value of qPCR with a glass slide inserted was 19. On the other hand, the Ct value of qPCR without a glass slide inserted was 22. A lower Ct value indicates better sensitivity. Each Ct value of 1 indicates a two-fold difference in sensitivity. In other words, it can be seen that the sensitivity is approximately eight times higher when a glass slide is inserted than when a glass slide is not inserted. Thus, differences in PCR results were observed depending on whether or not a glass slide was inserted. If bubbles were to form, an air layer would be generated in the chamber 10, causing an unstable reduction in the area of ​​the sample where a fluorescent reaction could be observed, which could lead to variations in fluorescence detection by the optical measurement unit 6X. On the other hand, inserting a glass slide is thought to suppress bubble formation, thereby improving the fluorescence detection capabilities of the optical measurement unit 6X.

[0035] (H) As shown in Figure 2B, the first member 12 and the second member 13 are the same member and have the same hardness, but are not limited to this. For example, the first member 12 may be harder than the second member 13. For the same reason as (G) above, this can provide the effects of suppressing expansion of the chamber 10 and suppressing the generation of bubbles, as well as the effects of improving heat transfer efficiency.

[0036] (I) FIG. 7 is a cross-sectional view corresponding to FIG. 4 according to a modified example. As shown in FIG. 7, the heat transfer surface 51 may have grooves 51a for allowing air to escape between the reaction vessel 1 and the heat transfer surface 51. This allows the air between the reaction vessel 1 and the heat transfer surface 51 to be removed, thereby preventing deterioration of heat transfer efficiency due to air. In the example of FIG. 7, three grooves 51a are formed, but the number of grooves 51a can be changed as appropriate. In the example of FIG. 7, the grooves 51a open at the ends of the heat transfer surface 51 (heat transfer member 52), but they do not have to open at the ends of the heat transfer surface 51 (heat transfer member 52). Furthermore, although the grooves 51a are straight grooves, this is not a limitation. For example, they may be curved grooves, straight grooves, or a combination thereof. The width of the grooves 51a may be any width that allows air to escape. Since increasing the width beyond a certain value does not provide any further improvement in the air escape effect, the width of the grooves 51a is preferably 1.0 mm or less. This is because if the width of the groove 51a is too wide, the area of ​​the heat transfer surface 51 that comes into contact with the reaction vessel 1 will be reduced.

[0037] (J) There is a response delay between the temperature of the test solution in the chamber 10 and the temperature of the heat transfer member 52 (heat transfer surface 51) detected by the temperature detection unit 56. In order to increase the speed at which the control temperature is switched in consideration of this temperature response delay, it is preferable to perform overshoot control as shown in FIGS. FIG. 8 is an explanatory diagram showing an example of overshoot control during temperature rise according to a modified example. As shown in FIG. 8, the temperature adjustment unit 5X can execute control to adjust the target temperature of the heat transfer surface 51 to one of multiple target temperatures, including a first target temperature T1 and a second target temperature T2. The second target temperature T2 is higher than the first target temperature T1. When changing the target temperature of the heat transfer surface 51 from the first target temperature T1 to the second target temperature T2, the temperature adjustment unit 5X controls the target temperature of the heat transfer surface 51 to a predetermined temperature (T2 + α) higher than the second target temperature T2 at a first time point S1. The value of α is, for example, 1 to 3°C, but is not limited thereto and can be any value. The time period during which the temperature is controlled to the predetermined temperature (T2 + α) is a predetermined time (S2 - S1), which is set in advance based on the volume of the chamber 10, the difference between the first target temperature T1 and the second target temperature T2, and the like. At a second time point S2 after control at the predetermined temperature (T2+α) has been performed for a predetermined time (S2-S1), the target temperature of the heat transfer surface 51 is controlled to a second target temperature T2. In this way, by controlling the target temperature of the heat transfer surface 51 to a predetermined temperature (T2+α) higher than the original second target temperature T2, the time required to raise the temperature of the test solution in the chamber 10 to the second target temperature T2 can be shortened compared to when the target temperature is simply controlled to the second target temperature T2. FIG. 9 is an explanatory diagram showing an example of overshoot control during temperature reduction according to a modified example. As shown in FIG. 9, the temperature adjustment unit 5X can execute control to adjust the target temperature of the heat transfer surface 51 to one of multiple target temperatures, including a first target temperature T1 and a second target temperature T2. The second target temperature T2 is lower than the first target temperature T1. When changing the target temperature of the heat transfer surface 51 from the first target temperature T1 to the second target temperature T2, the temperature adjustment unit 5X controls the target temperature of the heat transfer surface 51 to a predetermined temperature (T2-β) lower than the second target temperature T2 at a first time point S1. The value of β is, for example, 1 to 3°C, but is not limited thereto and can be any value. The time period during which the temperature is controlled to the predetermined temperature (T2-β) is a predetermined time (S2-S1), which is set in advance based on the volume of the chamber 10, the difference between the first target temperature T1 and the second target temperature T2, and the like. At a second time point S2 after control at the predetermined temperature (T2-β) has been performed for a predetermined time (S2-S1), the target temperature of the heat transfer surface 51 is controlled to a second target temperature T2. In this way, by controlling the target temperature of the heat transfer surface 51 to a predetermined temperature (T2-β) that is lower than the original second target temperature T2, the time required to raise the temperature of the test solution in the chamber 10 to the second target temperature T2 can be shortened compared to simply controlling the target temperature to the second target temperature T2.

[0038] (K) In the first embodiment, the fan 55 constantly rotates at a constant speed during operation of the PCR device, but this is not limiting. For example, in a structure including a heat transfer member 52 that can come into contact with the reaction vessel 1, a Peltier element 53 whose surface on the first side Y1 is thermally connected to the heat transfer member 52, a heat sink 54 whose surface on the second side Y2 of the Peltier element 53 is thermally connected, a fan 55 that cools the heat sink 54, and a processor that controls the Peltier element 53 and the fan 55, the processor may be configured to be able to execute at least one of a first mode, a second mode, a third mode, a fourth mode, and a fifth mode. The first mode is a mode in which a current for cooling the heat transfer member 52 is passed through the Peltier element 53 and the fan 55 is driven. The second mode is a mode in which a current for cooling the heat transfer member 52 is passed through the Peltier element 53, and the fan 55 is not driven. The third mode is a mode in which a current for heating the heat transfer member 52 is passed through the Peltier element 53, and the fan 55 is driven. The fourth mode is a mode in which a current for heating the heat transfer member 52 is passed through the Peltier element 53, and the fan 55 is not driven. The fifth mode is a mode in which the Peltier element 53 is not driven and the fan 55 is driven. In the first, third, and fifth modes, the fan 55 may be rotated at a constant speed. In the first and third modes, the rotation speed of the fan 55 may be changed according to the amount of current flowing through the Peltier element 53. In the fifth mode, the rotation speed of the fan 55 may be changed according to the temperature detected by the temperature detector 56.

[0039] (L) In the first embodiment, the storage space SP1 included in the measurement case 60 is a sealed space, but is not limited to this. For example, the storage space SP1 may have an openable and closable valve body other than the light conducting port 63, and the valve body may be in a closed state when the heat transfer surface 51 (reaction vessel 1) is heated and in an open state when the heat transfer surface 51 (reaction vessel 1) is cooled.

[0040] (M) In the first embodiment, the temperature detector 56 is provided to detect the temperature of the heat transfer member 52 that can come into contact with the reaction vessel 1, but this is not limiting. For example, the reaction vessel 1 may be provided with a sensor that detects the temperature inside the chamber 10, and the detection result of the sensor may be wirelessly transmitted to the processor.

[0041] (N) In the first embodiment, the first temperature and the second temperature are considered as one set, and one set of temperature control can be repeatedly performed, but this is not limited to this. For example, the temperatures indicating one set of elongation states may separately include a third temperature (65°C to 80°C). A fourth temperature (40°C to 50°C) for realizing a reverse transcription (RT) state may be performed once before the above repetition, and then one set consisting of the first and second temperatures may be repeated multiple times. The fourth temperature (40°C to 50°C) for realizing the reverse transcription (RT) state may be performed once, and then one set consisting of the first temperature, second temperature, and third temperature may be repeatedly performed multiple times.

[0042] [1] As described above, although not particularly limited, as in the above embodiment, the PCR device comprises a holding section 2 capable of holding a sheet-like reaction vessel 1 having a chamber 10 in which a test liquid is contained, a temperature adjustment section 5X capable of adjusting the temperature of the test liquid in the chamber 10 of the reaction vessel 1 held in the holding section 2 to a plurality of temperatures, and an optical measurement section 6X capable of measuring the fluorescence characteristics of the test liquid in the chamber 10, and at least a portion of the temperature adjustment range of the test liquid in the chamber 10 by the temperature adjustment section 5X may overlap with at least a portion of the measurement range of the test liquid in the chamber 10 by the optical measurement section 6X.

[0043] According to this configuration, the temperature adjustment unit 5X can adjust the temperature of the test solution in the chamber 10 to multiple temperatures, so the temperature can be raised and lowered within the chamber 10, making it possible to miniaturize the device, compared to a configuration in which the test solution is moved back and forth between a high-temperature heating unit and a low-temperature heating unit, or a configuration in which the chamber itself is moved between a high-temperature heating unit and a low-temperature heating unit. Furthermore, because at least a portion of the test solution temperature adjustment range overlaps with at least a portion of the test solution measurement range, it is possible to measure the fluorescence characteristics while adjusting the temperature of the test solution. This makes it possible to provide a PCR device with a simple configuration that can perform heating, cooling, and measurement in one location.

[0044] [2] In the PCR device described in [1] above, the optical measurement unit 6X may be arranged on a first side Y1 in the thickness direction (Y) of the sheet-shaped reaction vessel 1 when held in the holding unit 2, and the temperature adjustment unit 5X may be arranged on a second side Y2 opposite to the first side Y1 in the thickness direction (Y) of the sheet-shaped reaction vessel 1 when held in the holding unit 2. This is a suitable example that makes it possible to realize that at least a part of the temperature adjustment range of the test liquid and at least a part of the measurement range of the test liquid overlap.

[0045] [3] In the PCR device described in [2] above, the holding unit 2 may have a slit 20 into which a sheet-shaped reaction vessel 1 can be inserted, the wall surface of the second side Y2 forming the slit 20 has a heat transfer surface 51 that can come into contact with the reaction vessel 1, the temperature adjustment unit 5X can adjust the temperature of the test solution by heating or cooling the heat transfer surface 51, and the wall surface 21 of the first side Y1 forming the slit 20 has an optical guide port 63 that passes light from the chamber 10 to the optical measurement unit 6X. According to this configuration, the reaction vessel 1 is inserted into the slit 20, so that even if the reaction vessel 1 tries to warp or expand when heated, it can be held down by the wall surface that forms the slit 20, thereby preventing extreme deformation of the reaction vessel 1.

[0046] [4] In the PCR device described in [3] above, the heat transfer surface 51 may have a groove 51a for allowing air between the reaction vessel 1 and the heat transfer surface 51 to escape. If air exists between the reaction vessel 1 and the heat transfer surface 51, the degree of contact between the reaction vessel 1 and the heat transfer surface 51 will decrease. Grooves 51a for letting air escape are formed in the heat transfer surface 51, thereby improving the degree of contact between the reaction vessel 1 and the heat transfer surface 51. In particular, the internal pressure of the chamber 10 during heating tends to improve the degree of contact between the reaction vessel 1 and the heat transfer surface 51.

[0047] [5] The PCR device described in [3] above may be provided with a storage space SP1 that stores the optical measurement unit 6X, and the storage space SP1 may be an enclosed space that is not connected to the outside except for the optical conducting port 63. This can suppress or prevent the intrusion of ambient light, thereby avoiding a deterioration in the measurement accuracy of the optical measurement unit 6X. In addition, since the accommodation space SP1 has an airtight structure, it has a heat-retaining structure that makes it difficult for heated air to escape, and it is possible to shorten the time it takes to reach the target temperature in temperature control by the temperature adjustment unit 5X.

[0048] [6] The PCR device described in the above [3] may further include a transparent plate 7 disposed between the light conducting port 63 and the reaction vessel 1, and the transparent plate 7 may be harder than the reaction vessel 1. This configuration makes it possible to obtain the effect of suppressing expansion of the chamber 10 and the effect of pressing the reaction vessel 1 against the heat transfer surface 51 to increase the heat transfer efficiency.

[0049] [7] In the PCR apparatus described in [3] above, the reaction vessel 1 has a first member 12 that forms the chamber wall surface on the first side Y1 that forms the chamber 10, and a second member 13 that forms the chamber wall surface on the second side Y2 that forms the chamber 10, and the first member 12 may be harder than the second member 13. This configuration makes it possible to obtain the effect of suppressing expansion of the chamber 10 and the effect of pressing the reaction vessel 1 against the heat transfer surface 51 to increase the heat transfer efficiency.

[0050] [8] The PCR device according to any one of [1] to [3] above may be provided with a heat transfer surface 51 that can come into contact with the reaction vessel 1, and the temperature adjustment unit 5X is capable of controlling the temperature of the heat transfer surface 51 to one of a plurality of temperatures including a first target temperature T1 and a second target temperature T2 that is higher than the first target temperature T1, and when changing the temperature of the heat transfer surface 51 from the first target temperature T1 to the second target temperature T2, the temperature of the heat transfer surface 51 may be controlled to a predetermined temperature (T2+α) higher than the second target temperature T2 at a first time point S1, and may be controlled to the second target temperature T2 at a second time point S2 that is later than the first time point S1. This control allows for the target temperature to be switched earlier, taking into consideration the response delay between the temperature of the sample liquid in the chamber 10 and the temperature of the heat transfer surface 51.

[0051] [9] The PCR device according to any one of [1] to [3] above may be provided with a heat transfer surface 51 that can come into contact with the reaction vessel 1, and the temperature adjustment unit 5X is capable of executing control to adjust the target temperature of the heat transfer surface 51 to one of a plurality of target temperatures including a first target temperature T1 and a second target temperature T2 that is lower than the first target temperature T1, and when changing the target temperature of the heat transfer surface 51 from the first target temperature T1 to the second target temperature T2, the target temperature of the heat transfer surface 51 may be controlled to a predetermined temperature (T2-β) lower than the second target temperature T2 at a first time point S1, and may be controlled to the second target temperature T2 at a second time point S2 that is later than the first time point S1. This control allows for the target temperature to be switched earlier, taking into consideration the response delay between the temperature of the sample liquid in the chamber 10 and the temperature of the heat transfer surface 51.

[0052] Although the embodiments of the present disclosure have been described above with reference to the drawings, the specific configurations should not be considered to be limited to these embodiments. The scope of the present disclosure is defined not only by the description of the above embodiments but also by the claims, and further includes all modifications within the meaning and scope of the claims.

[0053] The structures employed in the above-described embodiments can be employed in any other embodiment. The specific configurations of the components are not limited to the above-described embodiments, and various modifications are possible within the scope of the present disclosure. [Explanation of symbols]

[0054] 1: Reaction vessel 2: Holding part 5X: Temperature adjustment section 6X: Optical measurement section 7: Transparent plate 10: Chamber 12: First member 13: Second member 20: Slit 21: Wall 22: Wall 51: Heat transfer surface 51a: Groove 63: Light conducting port S1: First time point S2: Second time point SP1: Containment Space T1: 1st target temperature T2: 2nd target temperature Y: Thickness direction Y1: First side Y2: Second side

Claims

1. a holder capable of holding a sheet-like reaction vessel having a chamber in which a test solution is contained; a temperature adjusting unit capable of adjusting the temperature of the reagent solution in the chamber of the reaction vessel held in the holding unit to a plurality of temperatures; an optical measurement unit capable of measuring the fluorescence characteristics of the test solution in the chamber; A PCR device, wherein at least a part of a temperature adjustment range of the test liquid in the chamber by the temperature adjustment unit overlaps with at least a part of a measurement range of the test liquid in the chamber by the optical measurement unit.

2. the optical measurement unit is disposed on a first side in a thickness direction of the sheet-like reaction vessel held by the holding unit, 2. The PCR device according to claim 1, wherein the temperature adjusting unit is disposed on a second side opposite to the first side in the thickness direction of the sheet-like reaction vessel held in the holding unit.

3. the holding part has a slit into which the sheet-like reaction vessel can be inserted, the second wall surface forming the slit has a heat transfer surface that can come into contact with the reaction vessel, and the temperature adjustment unit can adjust the temperature of the reagent solution by heating or cooling the heat transfer surface; The PCR device according to claim 2 , wherein the wall surface on the first side that forms the slit has a light guide port through which light passes from the chamber to the optical measurement unit.

4. The PCR device according to claim 3 , wherein the heat transfer surface has a groove for allowing air to escape between the reaction vessel and the heat transfer surface.

5. an accommodation space for accommodating the optical measurement unit; The PCR device according to claim 3 , wherein the accommodation space is a sealed space that does not communicate with the outside except for the light guide port.

6. a transparent plate disposed between the light guide port and the reaction vessel; The PCR device according to claim 3 , wherein the transparent plate is harder than the reaction vessel.

7. the reaction vessel has a first member forming a chamber wall surface on the first side that forms the chamber, and a second member forming a chamber wall surface on the second side that forms the chamber, The PCR device according to claim 3 , wherein the first member is harder than the second member.

8. a heat transfer surface that can come into contact with the reaction vessel; the temperature adjustment unit is capable of executing control to adjust the target temperature of the heat transfer surface to one of a plurality of target temperatures including a first target temperature and a second target temperature higher than the first target temperature; The PCR device according to any one of claims 1 to 3, wherein when changing the temperature of the heat transfer surface from the first target temperature to the second target temperature, the temperature of the heat transfer surface can be controlled to a predetermined temperature higher than the second target temperature at a first time point, and the temperature of the heat transfer surface can be controlled to the second target temperature at a second time point after the first time point.

9. a heat transfer surface that can come into contact with the reaction vessel; the temperature adjustment unit is capable of executing control to adjust the temperature of the heat transfer surface to one of a plurality of temperatures including a first target temperature and a second target temperature lower than the first target temperature; The PCR device according to any one of claims 1 to 3, wherein when changing the temperature of the heat transfer surface from the first target temperature to the second target temperature, the temperature of the heat transfer surface can be controlled to a predetermined temperature lower than the second target temperature at a first time point, and the temperature of the heat transfer surface can be controlled to the second target temperature at a second time point after the first time point.

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