JP6090554B2 - Heat cycle equipment - Google Patents

Description

  The present invention relates to a heat cycle apparatus.

  In recent years, gene-based medical care such as gene diagnosis and gene therapy has attracted attention as a result of the development of gene utilization technology, and in the field of agriculture and livestock, many methods using genes for variety discrimination and variety improvement have been developed. . Techniques such as PCR (Polymerase Chain Reaction) are widely used as techniques for utilizing genes. Today, PCR has become an indispensable technique for elucidating information on biological materials.

  The PCR method is a technique for amplifying a target nucleic acid by subjecting a solution (reaction solution) containing a nucleic acid (target nucleic acid) to be amplified and a reagent to thermal cycling. The thermal cycle is a process in which two or more stages of temperature are periodically applied to the reaction solution. In the PCR method, a method of performing a two-stage or three-stage thermal cycle is common.

  In the PCR method, a container for performing a biochemical reaction, generally called a tube or a biological sample reaction chip (biochip), is used. However, in the conventional method, there are problems that a large amount of reagents and the like are required, and that the apparatus becomes complicated in order to realize a thermal cycle necessary for the reaction, and that the reaction takes time. Therefore, a biochip and a reaction apparatus are required for performing PCR accurately and in a short time using a very small amount of reagent or specimen.

  In order to solve such a problem, Patent Document 1 discloses that a biological sample reaction chip filled with a reaction solution and a liquid that is not mixed with the reaction solution and has a specific gravity smaller than that of the reaction solution is rotated in the horizontal direction. A biological sample reaction apparatus is disclosed in which a reaction solution is moved by rotating around an axis to perform a thermal cycle.

JP 2009-136250 A

  The apparatus described in Patent Document 1 can perform a heat cycle (temperature cycle) in a short time. However, Patent Document 1 does not describe specific conditions for rotating the biological sample reaction chip, and it may take time to move the reaction solution. In order to perform the heat cycle in a shorter time, it was necessary to further reduce the time required for the reaction solution to move.

  The present invention has been made in view of the above problems, and according to some aspects of the present invention, it is possible to provide a thermal cycle apparatus capable of performing a thermal cycle in a short time.

The thermal cycle apparatus according to the application example is filled with a reaction liquid and a liquid having a specific gravity different from that of the reaction liquid and immiscible with the reaction liquid, and the reaction liquid moves in proximity to opposing inner walls. A mounting portion for mounting a reaction container including a flow path, and a temperature gradient forming that forms a temperature gradient in the direction in which the reaction solution moves with respect to the flow path when the reaction container is mounted in the mounting portion. And the mounting portion and the temperature gradient forming portion have a component perpendicular to the direction in which gravity acts, and when the reaction vessel is mounted on the mounting portion, the flow path is connected to the reaction liquid. A drive mechanism that rotates on a rotating shaft having a component perpendicular to the direction in which the actuator moves, and a control unit that controls the drive mechanism, wherein the control unit has a rotation speed of 0.5 rotation / Sec or more and acts on the reaction solution The magnitude of the centrifugal force for controlling the drive mechanism to be less than 1 times the force of gravity, a thermal cycler.

  According to this application example, the rotation axis has a component perpendicular to the direction in which gravity acts, and the direction in which the reaction solution moves through the flow path of the reaction vessel when the reaction vessel is attached to the attachment portion. Since the drive mechanism rotates the mounting part, the position of the lowest point or the highest point in the direction of gravity in the flow path of the reaction vessel mounted on the mounting part changes. To do. As a result, the reaction solution moves in the flow path where the temperature gradient is formed by the temperature gradient forming unit. Therefore, a thermal cycle can be applied to the reaction solution. Further, the drive mechanism can hold the reaction liquid at a predetermined temperature while holding the mounting portion and the temperature gradient forming portion in a predetermined arrangement. Therefore, it is possible to realize a heat cycle device that can easily control the heating time of the reaction solution. In addition, the control unit controls the drive mechanism so that the rotational speed of the drive mechanism is 0.5 rotations / second or more and the magnitude of the centrifugal force acting on the reaction liquid is not more than 1 times the gravity. As a result, the time from when the drive mechanism starts to rotate until the reaction solution starts to move is shortened. Therefore, it is possible to realize a heat cycle apparatus that can perform a heat cycle in a shorter time.

The perspective view of the heat cycle apparatus 1 which concerns on embodiment. The disassembled perspective view of the main body 10 of the heat cycle apparatus 1 which concerns on embodiment. FIG. 2 is a cross-sectional view schematically showing a cross section in a plane that passes through the line AA in FIG. Sectional drawing showing the structure of the reaction container 100 with which the thermal cycle apparatus 1 which concerns on embodiment is mounted | worn. The functional block diagram of the heat cycle apparatus 1 which concerns on embodiment. 6A is a cross-sectional view schematically showing a cross section in a plane perpendicular to the rotation axis R through the line AA in FIG. 1A in the first arrangement, and FIG. 6B is a second view. Sectional drawing which shows typically the cross section in the plane which passes along the AA line | wire of FIG. The flowchart for demonstrating the specific example of the control method of the heat cycle apparatus 1 which concerns on embodiment. The table | surface which shows the experimental result in 1st Example. The graph which shows the experimental result in 1st Example. The table | surface which shows the composition of the reaction liquid 140 in 2nd Example. Forward primer (F primer), reverse primer (R primer) and probe corresponding to influenza A (InfA), swine influenza A (SW InfA), swine influenza H1 (SW H1) and ribonuclease P (RNase P) The table | surface which shows the base sequence of Probe). The graph which shows the relationship between the cycle number of the heat cycle process in 2nd Example, and the measured brightness | luminance.

  DESCRIPTION OF EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. The embodiments described below do not unduly limit the contents of the present invention described in the claims. Also, not all of the configurations described below are essential constituent requirements of the present invention. The drawings are for convenience of explanation.

1. FIG. 1 is a perspective view of a heat cycle device 1 according to the present embodiment. FIG. 2 is an exploded perspective view of the main body 10 of the heat cycle apparatus 1 according to the present embodiment. FIG. 3 is a cross-sectional view schematically showing a cross section in a plane that passes through the line AA in FIG. 1 and is perpendicular to the rotation axis R. In FIG. 3, an arrow g represents the direction in which gravity acts.

  In the heat cycle apparatus 1 according to this embodiment, the reaction liquid 140 and the reaction liquid 140 are filled with a liquid 130 having a specific gravity different from that of the reaction liquid 140 and immiscible with the reaction liquid 140, and the reaction liquid 140 is close to the opposing inner walls. A mounting portion 15 for mounting the reaction vessel 100 including the moving flow path 110 (details will be described later in the section “2. Configuration of the reaction vessel mounted on the thermal cycler according to the present embodiment”); When the reaction vessel 100 is mounted on the mounting unit 15, the temperature gradient forming unit 20 that forms a temperature gradient with respect to the flow path 110 in the direction in which the reaction solution 140 moves, and the mounting unit 15 and the temperature gradient forming unit 20. And a component perpendicular to the direction in which the reaction solution 140 moves through the flow path 110 when the reaction vessel 100 is attached to the attachment portion 15. Rotate on rotation axis R To include a drive mechanism 30, a control unit 40 for controlling the drive mechanism 30.

  In the example shown in FIG. 1, the heat cycle apparatus 1 includes a main body 10 and a drive mechanism 30. As shown in FIG. 2, the main body 10 includes a mounting portion 15 and a temperature gradient forming portion 20. In the example shown in FIGS. 1 and 2, the temperature gradient forming unit 20 includes a first heating unit 21 and a second heating unit 22.

  The mounting portion 15 has a structure for mounting the reaction vessel 100. In the example shown in FIGS. 1 and 2, the mounting portion 15 of the heat cycle apparatus 1 has an insertion port 151 and has a slot structure in which the reaction vessel 100 is inserted through the insertion port 151. In the example shown in FIG. 2, the mounting unit 15 has a structure in which the reaction vessel 100 is inserted into a hole that penetrates the first heat block 21 b of the first heating unit 21 and the second heat block 22 b of the second heating unit 22. Yes. The first heat block 21b and the second heat block 22b will be described later. There may be a plurality of mounting parts 15 provided in the main body 10, and in the example shown in FIGS. 1 and 2, ten mounting parts 15 are provided in the main body 10. In the example shown in FIGS. 2 and 3, the mounting unit 15 is configured as a part of the first heating unit 21 and the second heating unit 22. As long as the relationship does not change, the mounting unit 15, the first heating unit 21, and the second heating unit 22 may be configured as separate members.

  In the present embodiment, the mounting portion 15 has a slot structure, but the mounting portion 15 may have any structure that can hold the reaction vessel 100. For example, a structure in which the reaction container 100 is fitted in a recess that matches the shape of the reaction container 100, or a structure in which the reaction container 100 is held therebetween may be employed.

  The temperature gradient forming unit 20 forms a temperature gradient in the direction in which the reaction solution 140 moves with respect to the flow path 110 when the reaction vessel 100 is mounted on the mounting unit 15. Here, “forming a temperature gradient” means forming a state in which the temperature changes along a predetermined direction. Therefore, “to form a temperature gradient in the direction in which the reaction liquid 140 moves” means to form a state in which the temperature changes along the direction in which the reaction liquid 140 moves. “The state in which the temperature changes along the predetermined direction” is, for example, that the temperature may be monotonously high or low along the predetermined direction, or from a change in which the temperature increases along the predetermined direction. You may change on the way from the change which becomes low, or from the change which becomes low to the change which becomes high.

  In the main body 10 of the heat cycle apparatus 1 shown in FIGS. 1 to 3, the first heating unit 21 of the temperature gradient forming unit 20 is relatively far from the insertion port 151 of the mounting unit 15. The second heating unit 22 is disposed on the side closer to the insertion port 151 of the mounting unit 15.

The first heating unit 21 heats the first region 111 of the flow path 110 of the reaction container 100 when the reaction container 100 is mounted on the mounting unit 15. In the example shown in FIG. 3, the first heating unit 21 is disposed in the main body 10 at a position for heating the first region 111 of the reaction vessel 100.

  The first heating unit 21 may include a mechanism that generates heat and a member that transmits the generated heat to the reaction vessel 100. In the example shown in FIG. 2, the first heating unit 21 includes a first heater 21 a as a mechanism for generating heat and a first heat block 21 b as a member for transmitting the generated heat to the reaction vessel 100. ing.

  In the heat cycle apparatus 1, the first heater 21 a is a cartridge heater, and is connected to an external power source (not shown) by a conducting wire 19. The first heater 21a is not limited to this, and a carbon heater, a sheet heater, an IH heater (electromagnetic induction heater), a Peltier element, a heating liquid, a heating gas, and the like can be used. The first heater 21a is inserted into the first heat block 21b, and the first heat block 21b is heated when the first heater 21a generates heat. The first heat block 21 b is a member that transfers heat generated from the first heater 21 a to the reaction vessel 100. In the heat cycle apparatus 1, the first heat block 21b is an aluminum block. Since the temperature control of the cartridge heater is easy, the temperature of the first heating unit 21 can be easily stabilized by using the first heater 21a as a cartridge heater. Therefore, a more accurate thermal cycle can be realized.

  The material of the heat block can be appropriately selected in consideration of conditions such as thermal conductivity, heat retention and ease of processing. For example, since aluminum has high thermal conductivity, the reaction vessel 100 can be efficiently heated by making the first heat block 21b aluminum. In addition, since heating unevenness hardly occurs in the heat block, a highly accurate thermal cycle can be realized. Further, since the processing is easy, the first heat block 21b can be accurately molded, and the heating accuracy can be improved. Therefore, a more accurate thermal cycle can be realized. In addition, as a material of the heat block, for example, a copper alloy may be used, or a plurality of materials may be combined.

  The first heating unit 21 is preferably in contact with the reaction vessel 100 when the reaction vessel 100 is attached to the attachment unit 15. As a result, when the reaction vessel 100 is heated by the first heating unit 21, the heat of the first heating unit 21 is more stably transmitted to the reaction vessel 100 than in a configuration in which the first heating unit 21 and the reaction vessel 100 do not contact each other. Therefore, the temperature of the reaction vessel 100 can be stabilized. When the mounting part 15 is formed as a part of the first heating unit 21 as in the present embodiment, it is preferable that the mounting part 15 contacts the reaction vessel 100. Thereby, the heat of the first heating unit 21 can be stably transmitted to the reaction vessel 100, so that the reaction vessel 100 can be efficiently heated.

  When the reaction vessel 100 is attached to the attachment unit 15, the second heating unit 22 sets the second region 112 of the flow path 110 of the reaction vessel 100 closer to the insertion port 151 than the first region 111 to the first temperature. Are heated to a different second temperature. In the example shown in FIG. 3, the second heating unit 22 is disposed in the main body 10 at a position for heating the second region 112 of the reaction vessel 100. The second heating unit 22 includes a second heater 22a and a second heat block 22b. The configuration of the second heating unit 22 in the present embodiment is the same as that of the first heating unit 21 except that the region of the reaction vessel 100 to be heated and the heating temperature are different from those of the first heating unit 21. In addition, you may employ | adopt a heating mechanism from which the 1st heating part 21 and the 2nd heating part 22 differ. Further, the first heat block 21b and the second heat block 22b may be made of different materials.

The first heating unit 21 and the second heating unit 22 are provided in the main body 10 so as to be separated from each other. As a result, the first heating unit 21 and the second heating unit 22 that are controlled to different temperatures are less likely to affect each other, and the temperatures of the first heating unit 21 and the second heating unit 22 are easily stabilized. A spacer may be provided between the first heating unit 21 and the second heating unit 22. In the main body 10 of the heat cycle apparatus 1, the first heating unit 21 and the second heating unit 22 are fixed around the fixing member 16, the flange 17, and the flange 18. The flange 18 is supported by a bearing 31. In addition, as long as a temperature gradient is formed to such an extent that desired reaction accuracy can be ensured, the number of heating units may be any number of 2 or more.

  At least one of the first heating unit 21 and the second heating unit 22 may have a plurality of modes (operation modes) having different temperatures. Thereby, it is possible to realize the thermal cycle apparatus 1 that can realize various thermal cycles such as a plurality of thermal cycles with different temperature conditions and a thermal cycle in which the temperature conditions are changed midway.

  The temperature of the 1st heating part 21 and the 2nd heating part 22 is controlled by the temperature sensor which is not shown in figure and the control part 40 mentioned later. It is preferable that the temperature of the 1st heating part 21 and the 2nd heating part 22 is set so that the reaction container 100 may be heated to desired temperature. Details of the temperature control of the first heating unit 21 and the second heating unit 22 will be described in detail in the section “3. Control Example of Thermal Cycler”. In addition, the temperature of the 1st heating part 21 and the 2nd heating part 22 should just be controlled so that the 1st area | region 111 and the 2nd area | region 112 of the reaction container 100 may be heated to desired temperature. For example, by considering the material and size of the reaction vessel 100, the temperature of the first region 111 and the second region 112 can be heated to a desired temperature more accurately. In this embodiment, the temperature of the 1st heating part 21 and the 2nd heating part 22 is measured with a temperature sensor. The temperature sensor of this embodiment is a thermocouple. The temperature sensor is not limited to this, and for example, a resistance temperature detector or a thermistor may be used.

  The drive mechanism 30 has a component perpendicular to the direction in which gravity is applied to the mounting unit 15, the first heating unit 21, and the second heating unit 22, and the reaction vessel 100 is mounted on the mounting unit 15. The flow path 110 is rotated around a rotation axis R having a component perpendicular to the direction in which the reaction solution 140 moves. In the present embodiment, the drive mechanism 30 has the arrangement of the mounting unit 15 and the temperature gradient forming unit 20 in the first arrangement and the position of the lowest point in the flow path 110 in the direction in which gravity acts is the first. Rotate between a second arrangement different from the arrangement.

  The direction “having a component perpendicular to the direction in which gravity acts” is the vector sum of “component parallel to the direction in which gravity acts” and “component perpendicular to the direction in which gravity acts”. In this case, it is a direction having a component perpendicular to the direction in which gravity acts.

  The direction of “having a component perpendicular to the direction in which the reaction solution 140 moves through the flow path 110” refers to “the component parallel to the direction in which the reaction liquid 140 moves through the flow path 110” and “reaction of the flow path 110. This is a direction having a component perpendicular to the direction in which the reaction solution 140 moves in the flow path 110 in the case of the vector sum of “component perpendicular to the direction in which the solution 140 moves”.

  In the heat cycle apparatus 1 according to the first embodiment, the drive mechanism 30 rotates the mounting unit 15, the first heating unit 21, and the second heating unit 22 around the same rotation axis R. In the present embodiment, the drive mechanism 30 includes a motor and a drive shaft (not shown), and is configured by connecting the drive shaft and the flange 17 of the main body 10. When the motor of the drive mechanism 30 is operated, the main body 10 is rotated with the drive shaft as the rotation axis R. In the present embodiment, ten mounting portions 15 are provided along the direction of the rotation axis R. The drive mechanism 30 is not limited to a motor, and for example, a handle, a mainspring, or the like can be adopted.

The heat cycle apparatus 1 may include a control unit 40. The control unit 40 controls the drive mechanism 30. The control unit 40 may further control the temperature gradient forming unit 20 (the first heating unit 21 and the second heating unit 22). The control unit 40 may further control the measurement unit 50. An example of control by the control unit 40 will be described in detail in the section “3. Control example of heat cycle device”. Control unit 4
0 may be configured to be realized by a dedicated circuit and to perform control described later. The control unit 40 functions as a computer by, for example, a CPU (Central Processing Unit) executing a control program stored in a storage device such as a ROM (Read Only Memory) or a RAM (Random Access Memory), which will be described later. It may be configured to perform control. In this case, the storage device may have a work area for temporarily storing intermediate data and control results associated with the control. Moreover, the control part 40 may have a timer for measuring time. Moreover, the control part 40 may control the 1st heating part 21 and the 2nd heating part 22 to desired temperature based on the output of the temperature sensor which is not shown in figure mentioned above.

  The heat cycle apparatus 1 may include a measurement unit 50. The measurement unit 50 measures the intensity of light having a predetermined wavelength. In the present embodiment, a fluorescence detector is employed as the measurement unit 50. Thus, for example, by adding a fluorescent probe whose intensity of light of a predetermined wavelength changes by complementary binding to specific DNA in the reaction solution 140, it can be used for applications involving fluorescence measurement such as real-time PCR. The cycle device 1 can be used. The number of measurement units 50 is arbitrary as long as measurement can be performed without any problem. In the example shown in FIG. 1, fluorescence measurement is performed by moving one measuring unit 50 along a slide 52.

  The position of the measurement unit 50 is preferably on the side closer to the first heating unit 21 than on the side closer to the second heating unit 22. This makes it difficult to obstruct work when the reaction container 100 is mounted on the mounting portion 15. Moreover, the measurement part 50 may be provided so that the light from the area | region including the 1st area | region 111 of the reaction container 100 may be measured. When the temperature of the first heating unit 21 is set to PCR annealing and extension temperature (temperature at which the annealing and extension reaction proceeds), the intensity of light having a predetermined wavelength that correlates with the amount of specific DNA emitted by the fluorescent probe is measured. it can. Therefore, appropriate fluorescence measurement can be performed in real-time PCR. Further, when using the reaction vessel 100 with a lid (sealing part 120) described later, the first region 111 on the side farther from the lid than the second region 112 on the side closer to the lid, Since there are few members existing between the measurement unit 50 and the reaction solution 140, more appropriate fluorescence measurement can be performed.

  As described above, when the thermal cycle apparatus 1 is used for real-time PCR, the measurement unit 50 is provided on the side closer to the first heating unit 21 during the period in which the thermal cycle necessary for PCR is applied to the reaction solution 140. It is preferable to set the first heating unit 21 to PCR annealing and extension temperature (about 50 ° C. to 75 ° C.). In this case, the second heating unit 22 close to the insertion port 151 is set to a heat denaturation temperature (about 90 ° C. to 100 ° C.) higher than the annealing and extension temperature of PCR.

  The heat cycle apparatus 1 preferably includes a structure that holds the reaction vessel 100 at a predetermined position with respect to the first heating unit 21 and the second heating unit 22. Accordingly, a predetermined region of the reaction vessel 100 can be heated by the first heating unit 21 and the second heating unit 22. More specifically, the first region 111 of the flow path 110 constituting the reaction vessel 100 can be heated by the first heating unit 21, and the second region 112 can be heated by the second heating unit 22. In the present embodiment, the first heating unit 21 and the second heating unit are appropriately set by appropriately setting the sizes of the through holes (the diameter of the mounting unit 15) provided in the first heat block 21b and the second heat block 22b. The reaction vessel 100 is held at a predetermined position with respect to the portion 22.

  The first heat block 21b may have a structure having fins 210. As a result, the surface area of the first heating unit 21 is increased, and thus the time required for changing the temperature of the first heating unit 21 from a high temperature to a low temperature is shortened.

The heat cycle apparatus 1 may include a fan 500 that blows air to the first heating unit 21 and the second heating unit 22. By blowing air, the movement of heat between the first heating unit 21 and the second heating unit 22 can be suppressed. Therefore, the 1st heating part 21 controlled by mutually different temperature.
And the second heating unit 22 are less likely to affect each other, so that the temperatures of the first heating unit 21 and the second heating unit 22 are easily stabilized.

2. FIG. 4 is a cross-sectional view showing the configuration of the reaction vessel 100 attached to the thermal cycler 1 according to this embodiment. In FIG. 4, an arrow g represents the direction in which gravity acts.

  The reaction vessel 100 is filled with a reaction liquid 140 and a liquid 130 having a specific gravity different from that of the reaction liquid 140 and immiscible with the reaction liquid 140 (hereinafter referred to as “liquid 130”). It includes a flow path 110 that moves along the inner wall. In the present embodiment, the liquid 130 is a liquid having a specific gravity smaller than that of the reaction liquid 140 and immiscible with the reaction liquid 140. As the liquid 130, for example, a liquid that is not miscible with the reaction liquid 140 and has a higher specific gravity than the reaction liquid 140 may be employed. In the example shown in FIG. 4, the reaction vessel 100 includes a flow path 110 and a sealing portion 120. The flow path 110 is filled with the reaction liquid 140 and the liquid 130 and is sealed by the sealing portion 120.

  The channel 110 is formed so that the reaction solution 140 moves along the opposing inner walls. Here, the “opposite inner walls” of the flow channel 110 mean two regions of the wall surface of the flow channel 110 that are in a positional relationship facing each other. “Along” means a state in which the distance between the reaction solution 140 and the wall surface of the channel 110 is short, and includes a state in which the reaction solution 140 is in contact with the wall surface of the channel 110. Therefore, “the reaction solution 140 moves along the opposing inner walls” means that the reaction solution 140 is in a state in which the distance is close to both of the two regions on the wall surface of the flow path 110 that face each other. Means "move". In other words, the distance between the two inner walls facing each other in the flow path 110 is such a distance that the reaction solution 140 moves along the inner wall.

  When the flow path 110 of the reaction vessel 100 has such a shape, the direction in which the reaction liquid 140 moves in the flow path 110 can be regulated, so that the path through which the reaction liquid 140 moves in the flow path 110 can be defined to some extent. Thereby, the time required for the reaction liquid 140 to move in the flow path 110 can be limited to a certain range. Therefore, the distance between the two inner walls facing each other in the flow path 110 is desired to be a variation in the heat cycle conditions applied to the reaction liquid 140 caused by a variation in the time during which the reaction liquid 140 moves in the flow path 110. It is preferable that the accuracy of the reaction can be satisfied, that is, the result of the reaction can satisfy the desired accuracy. More specifically, the distance in the direction perpendicular to the direction in which the reaction solution 140 moves between two opposing inner walls of the flow path 110 is such that two or more droplets of the reaction solution 140 do not enter. Is desirable.

  In the example shown in FIG. 4, the outer shape of the reaction vessel 100 is a truncated cone, and a flow path 110 having a longitudinal direction in the direction along the central axis (the vertical direction in FIG. 4) is formed. The shape of the flow path 110 is a cross section in a direction perpendicular to the longitudinal direction of the flow path 110, that is, a cross section perpendicular to the direction in which the reaction solution 140 moves in a region of the flow path 110 (this is the cross section of the flow path 110). A “conical section” is a circular truncated cone shape. Therefore, in the reaction vessel 100, the opposed inner walls of the flow channel 110 are regions including two points on the wall surface of the opposed flow channel 110 across the center of the cross section of the flow channel 110. Further, “the direction in which the reaction liquid 140 moves” is the longitudinal direction of the flow path 110.

The shape of the flow path 110 is not limited to a frustum shape, and may be a columnar shape, for example. In addition, the cross-sectional shape of the flow path 110 is not limited to a circle, but may be any shape such as a polygon or an ellipse as long as the reaction solution 140 can move along the opposing inner walls. For example, when the cross section of the flow path 110 of the reaction vessel 100 is polygonal, the “opposite inner wall” is the opposite of the flow path when the cross section inscribed in the flow path 110 is assumed to be a circular flow path. It shall be an inner wall. That is, the flow path 110 is moved so that the reaction solution 140 moves along the opposing inner walls of the virtual flow path inscribed in the flow path 110 and having a circular cross section.
Should just be formed. As a result, even when the cross section of the flow path 110 is polygonal, a path through which the reaction solution 140 moves between the first region 111 and the second region 112 can be defined to some extent. Therefore, the time required for the reaction solution 140 to move between the first region 111 and the second region 112 can be limited to a certain range.

  The first region 111 of the reaction vessel 100 is a partial region of the flow path 110 that is heated by the first heating unit 21. The second region 112 is a partial region of the flow path 110 that is heated by the second heating unit 22 and is different from the first region 111. In the example shown in FIG. 4, the first region 111 is a region including one end portion in the longitudinal direction of the flow path 110, and the second region 112 includes the other end portion in the longitudinal direction of the flow path 110. It is an area. In the example shown in FIG. 4, a region surrounded by a dotted line including an end portion relatively far from the sealing portion 120 in the flow channel 110 is the first region 111, and the sealing portion in the flow channel 110. A region surrounded by a dotted line including an end portion on the side relatively close to 120 is a second region 112. In the heat cycle apparatus 1 according to this embodiment, the first heating unit 21 heats the first region 111 of the reaction vessel 100 and the second heating unit 22 heats the second region 112 of the reaction vessel 100, thereby causing a reaction. A temperature gradient is formed in the direction in which the reaction solution 140 moves with respect to the flow path 110 of the container 100.

The flow path 110 is filled with the liquid 130 and the reaction liquid 140. Since the liquid 130 is immiscible with the reaction liquid 140, that is, does not mix, the reaction liquid 140 is held in the liquid 130 in the form of droplets as shown in FIG. Since the specific gravity of the reaction liquid 140 is larger than that of the liquid 130, the reaction liquid 140 is located in the lowermost region in the direction in which gravity acts in the flow path 110. As the liquid 130, for example, dimethyl silicone oil or paraffin oil can be used. The reaction liquid 140 is a liquid containing components necessary for the reaction. For example, when the reaction is PCR, the reaction solution 140 is complementarily bound to DNA to be amplified, a DNA polymerase (PCR enzyme) necessary for amplifying the DNA, a primer, and a specific DNA sequence. Thus, a fluorescent probe or the like in which the intensity of light of a predetermined wavelength (light emitted from the fluorescent probe) changes is included. For example, when the reaction is RT-PCR (Reverse Transcription Polymerase Chain Reaction), the reaction solution 140 contains reverse transcriptase, RNA as a template for reverse transcription, reverse transcribed cDNA (complementary deoxyribonucleic acid).
DNA polymerase (PCR enzyme) necessary for amplifying acid), a primer, a fluorescent probe in which the intensity of light of a predetermined wavelength is changed by complementary binding to a specific DNA sequence, and the like. For example, when PCR is performed using oil as the liquid 130, the reaction solution 140 is preferably an aqueous solution containing the above-described components.

3. Control Example of Thermal Cycle Device FIG. 5 is a functional block diagram of the thermal cycle device 1 according to the present embodiment. The control unit 40 controls the temperature of the first heating unit 21 by outputting a control signal S <b> 1 to the first heating unit 21. The control unit 40 controls the temperature of the second heating unit 22 by outputting a control signal S <b> 2 to the second heating unit 22. The control unit 40 controls the drive mechanism 30 by outputting a control signal S3 to the drive mechanism 30. The control unit 40 controls the measurement unit 50 by outputting a control signal S4 to the measurement unit 50.

  Next, a control example of the heat cycle apparatus 1 according to the present embodiment will be described. Hereinafter, when the mounting unit 15, the first heating unit 21, and the second heating unit 22 are arranged, when the reaction vessel 100 is mounted on the mounting unit 15, the position of the lowest point of the flow path 110 in the direction in which gravity acts. Example of control for rotating between the first arrangement in which the first region 111 is in the first region 111 and the second arrangement in which the position of the lowest point of the flow path 110 in the direction in which the gravity acts is in the second region 112 I will explain to you.

6A is a cross-sectional view schematically showing a cross section in a plane perpendicular to the rotation axis R through the line AA in FIG. 1A in the first arrangement, and FIG. A in FIG. 1A in the arrangement of 2
It is sectional drawing which shows typically the cross section in the surface which passes along the -A line and is perpendicular to the rotation axis R. 6A and 6B, the white arrow indicates the direction of rotation of the main body 10, and the arrow g indicates the direction in which gravity acts.

  As shown in FIG. 6A, the first arrangement is such that when the reaction vessel 100 is mounted on the mounting portion 15, the first region 111 is positioned at the lowermost portion of the flow path 110 in the direction in which gravity acts. It is arrangement to do. In the example shown in FIG. 6A, in the first arrangement, the reaction liquid 140 having a specific gravity greater than that of the liquid 130 exists in the first region 111. In addition, as shown in FIG. 6B, when the reaction vessel 100 is attached to the attachment portion 15, the second arrangement is the lowest part of the flow path 110 in the direction in which the second region 112 acts by gravity. It is the arrangement located in. In the example shown in FIG. 6B, in the second arrangement, the reaction liquid 140 having a specific gravity greater than that of the liquid 130 exists in the second region 112.

  Thus, the drive mechanism 30 rotates the mounting unit 15, the first heating unit 21, and the second heating unit 22 between the first arrangement and the second arrangement different from the first arrangement. Thus, the reaction solution 140 can be subjected to a heat cycle.

  According to this embodiment, the rotation axis R has a component perpendicular to the direction in which gravity acts, and reacts with the flow path 110 of the reaction vessel 100 when the reaction vessel 100 is attached to the attachment portion 15. Since the liquid 140 has a component perpendicular to the direction in which the liquid 140 moves, the direction in which gravity acts in the flow path 110 of the reaction vessel 100 mounted on the mounting unit 15 when the drive mechanism 30 rotates the mounting unit 15. The position of the lowest point or the highest point in is changed. As a result, the reaction solution 140 moves in the flow path 110 where the temperature gradient is formed by the temperature gradient forming unit 20. Accordingly, the reaction solution 140 can be subjected to a heat cycle.

  Further, the drive mechanism 30 can maintain the reaction solution 140 at a predetermined temperature while maintaining the mounting unit 15 and the temperature gradient forming unit 20 in a predetermined arrangement. For example, by switching the arrangement of the mounting unit 15, the first heating unit 21, and the second heating unit 22, the reaction vessel 100 is held in the first arrangement and the reaction vessel 100 is held in the second arrangement. The state can be switched. The first arrangement is an arrangement in which the first region 111 of the flow path 110 constituting the reaction vessel 100 is located at the lowermost part of the flow path 110 in the direction in which gravity acts. The second arrangement is an arrangement in which the second region 112 of the flow path 110 constituting the reaction vessel 100 is located at the lowermost part of the flow path 110 in the direction in which gravity acts. That is, when the specific gravity of the reaction liquid 140 is larger than that of the liquid 130, the reaction liquid 140 is placed in the first region 111 in the first arrangement and the reaction liquid 140 is put in the second arrangement in the second arrangement due to the action of gravity. It can be held in area 112. Since the first region 111 is heated by the first heating unit 21 and the second region 112 is heated by the second heating unit 22, the first region 111 and the second region 112 can have different temperatures. Therefore, since the reaction liquid 140 can be held at a predetermined temperature while holding the reaction vessel 100 in the first arrangement or the second arrangement, the thermal cycle apparatus 1 capable of easily controlling the heating time can be provided.

  In addition, the controller 40 drives the drive mechanism 30 so that the rotational speed of the drive mechanism 30 is 0.5 rotations / second or more and the magnitude of the centrifugal force acting on the reaction solution 140 is less than 1 times the gravity. To control. This shortens the time from when the drive mechanism 30 starts to rotate until the reaction solution 140 starts to move. If the time from when the drive mechanism 30 starts to rotate until the reaction solution 140 starts to move becomes short, the time that the drive mechanism 30 ends after the rotation has been completed can be set to be short. it can. Therefore, the heat cycle apparatus 1 that can perform the heat cycle in a short time can be realized. In addition, the effect that the thermal cycle can be performed in a short time will be described in detail in the section “4. Examples”.

The drive mechanism 30 is rotated in the opposite direction between the first arrangement and the first arrangement, and when the drive mechanism 30 is rotated from the second arrangement to the first arrangement. And the 2nd heating part 22 may be rotated. This eliminates the need for a mechanism for reducing the twisting of wiring such as the conductive wire 19 caused by rotation. Therefore, the heat cycle apparatus 1 suitable for downsizing can be realized. Further, the number of rotations when rotating from the first arrangement to the second arrangement and the number of rotations when rotating from the second arrangement to the first arrangement are less than one rotation (the rotation angle is 360 °). Less). This can reduce the degree to which the wiring is twisted. Further, as shown in FIGS. 1 and 2, the flange 18 may be configured to wind the conductive wire 19.

  The control unit 40 controls the first heating unit 21 to a first temperature and a second temperature different from the first temperature, and controls the second heating unit 22 to a third temperature different from the first temperature and the second temperature. May be.

  That is, the control unit 40 has a mode for controlling the first heating unit 21 to the first temperature and a mode for controlling the second heating unit 21 to the second temperature, and controls the second heating unit 22 to the third temperature. Since the first temperature, the second temperature, and the third temperature are different from each other, the first heating unit 21 and the second heating unit 22 can realize a thermal cycle in which at least three types of temperatures are combined.

  Next, with respect to a specific example of the control method of the heat cycle apparatus 1, when real-time measurement is performed by RT-PCR including a hot start step (a step of activating a hot start enzyme by heat in PCR using a hot start PCR enzyme) Will be described as an example. It is assumed that the reaction solution 140 contains a fluorescent probe that changes the intensity of light of a predetermined wavelength by complementary binding to specific DNA. FIG. 7 is a flowchart for explaining a specific example of the control method of the thermal cycler 1 according to this embodiment. Hereinafter, an example in which Step S100 is performed after mounting the reaction container 100 on the mounting unit 15 will be described, but the reaction container 100 may be mounted on the mounting unit 15 after Step S100.

  In this specific example, first, the control unit 40 controls the first heating unit 21 to the first temperature, and the arrangement of the mounting unit 15, the first heating unit 21, and the second heating unit 22 is the first arrangement and the first arrangement. When the time has elapsed, the first heating unit 21 is controlled to a second temperature higher than the first temperature, the second heating unit 22 is controlled to a third temperature higher than the second temperature, the mounting unit 15, A first process for controlling the drive mechanism 30 so as to switch the arrangement of the first heating unit 21 and the second heating unit 22 from the first arrangement to the second arrangement is performed.

  More specifically, first, the control unit 40 controls the temperature of the first heating unit 21 to the first temperature by outputting a control signal S1 to the first heating unit 21 (step S100). In this specific example, the first temperature is a temperature at which the reverse transcription reaction proceeds by reverse transcriptase. “Temperature at which reverse transcription reaction proceeds by reverse transcriptase” is a temperature that depends on the type of reverse transcriptase, but is generally in the range of 20 ° C. or higher and 70 ° C. or lower. In general, the reaction is likely to proceed within the following range. In this specific example, the placement of the mounting portion 15, the first heating portion 21, and the second heating portion 22 during the initial operation is the first placement. Accordingly, the reaction solution 140 is held in the first region 111. That is, the reaction liquid 140 is maintained at the first temperature.

  In step S100, the control unit 40 may control the temperature of the second heating unit 22 to a temperature at which the reverse transcriptase is not deactivated by outputting a control signal S2 to the second heating unit 22. . The “temperature at which the reverse transcriptase does not inactivate” is a temperature that depends on the type of reverse transcriptase, but is generally in the range of about 20 ° C. to 70 ° C. In general, at a temperature exceeding 70 ° C., reverse transcriptase tends to be inactivated or deteriorated. By controlling the temperature of the second heating unit 22 to a temperature at which the reverse transcriptase is not deactivated, the reaction solution 140 is brought to a temperature at which the reverse transcriptase is deactivated when the reaction vessel 100 is mounted on the mounting unit 15. It will not be exposed.

  After step S100, the control unit 40 determines whether or not the first time has elapsed after step S100 is completed (after the first heating unit 21 has reached the first temperature) (step S102). In this specific example, the first time is the time required for the reverse transcription reaction. If the control unit 40 determines that the first time has not elapsed (NO in step S102), the control unit 40 repeats step S102.

  When the control unit 40 determines that the first time has passed (YES in step S102), the control unit 40 outputs the control signal S1 to the first heating unit 21 to thereby perform the first heating. By controlling the temperature of the unit 21 to the second temperature and outputting the control signal S2 to the second heating unit 22, the temperature of the second heating unit 22 is controlled to the second temperature (step S104). In this example, the second temperature is the annealing and extension temperature in PCR. “Annealing and extension temperature in PCR” is a temperature that depends on the type of enzyme (DNA polymerase in this specific example) used to amplify a nucleic acid, but is generally in the range of about 50 ° C. to 70 ° C. Is within. In this specific example, the third temperature is a heat denaturation temperature in PCR. “Thermal denaturation temperature in PCR” is a temperature depending on the type of enzyme for amplifying nucleic acid, but is generally in the range of about 90 ° C. to 100 ° C.

  After step S104, the control unit 40 outputs the control signal S3 to the drive mechanism 30, thereby changing the arrangement of the mounting unit 15, the first heating unit 21, and the second heating unit 22 from the first arrangement to the second arrangement. The drive mechanism 30 is controlled to switch to the arrangement (step S106). As described above, since the placement of the mounting portion 15, the first heating portion 21, and the second heating portion 22 is the first placement until step S104, the mounting portion 15, the first heating portion is performed by performing step S102. The arrangement of the heating unit 21 and the second heating unit 22 is a second arrangement. When the placement of the mounting portion 15, the first heating portion 21, and the second heating portion 22 is the second placement, the reaction solution 140 is held in the second region 112. That is, the reaction liquid 140 is maintained at the third temperature.

  After step S106, the control unit 40 performs a second process in which the placement of the mounting unit 15, the first heating unit 21, and the second heating unit 22 is the second placement and the second time elapses.

  More specifically, after step S106, the control unit 40 determines whether or not the second time has elapsed since step S106 was completed (step S108). In this specific example, the second time is a time required for the activation (hot start) of the PCR enzyme. If the control unit 40 determines that the second time has not elapsed (NO in step S108), the control unit 40 repeats step S108.

  When the control unit 40 determines that the second time has elapsed (in the case of YES in step S108), the control unit 40 has the second arrangement of the mounting unit 15, the first heating unit 21, and the second heating unit 22 as the second. When the third time elapses in the arrangement, the control signal S3 is output to the drive mechanism 30 to change the arrangement of the mounting unit 15, the first heating unit 21, and the second heating unit 22 to the second arrangement. A third process for controlling the drive mechanism 30 so as to switch from the first to the first arrangement is performed.

More specifically, first, the control unit 40 determines whether or not the third time has elapsed since step S108 was completed (step S110). In this specific example, the third time is a time required for heat denaturation of DNA in PCR. When the control unit 40 determines that the third time has not elapsed (NO in step S110), the control unit 40 repeats step S110. When the control unit 40 determines that the third time has elapsed (in the case of YES in step S110), the control unit 40 outputs a control signal S3 to the drive mechanism 30, thereby causing the mounting unit 15, The drive mechanism 30 is controlled so that the arrangement of the first heating unit 21 and the second heating unit 22 is switched from the second arrangement to the first arrangement (step S112). Mounting part 15,
When the arrangement of the first heating unit 21 and the second heating unit 22 is the first arrangement, the reaction solution 140 is held in the first region 111. That is, as a result of step S112, the reaction solution 140 is held at the second temperature.

  According to this specific example, the temperature of the first heating unit 21 is different between the first process, the second process, and the third process. As described above, by performing the first process, the second process, and the third process with different temperature conditions, the reverse transcription reaction can be performed before the PCR, so that the thermal cycle apparatus 1 suitable for RT-PCR is realized. it can.

  Moreover, when performing PCR including a hot start process, the temperature which activates a PCR enzyme is comparable to heat denaturation temperature. Therefore, by performing the second treatment, it is possible to realize a thermal cycle including a hot start of PCR, which is a step of activating the PCR enzyme.

  In this specific example, after the third process, the control unit 40 outputs the control signal S4 to the measurement unit 50 to measure the intensity of light of a predetermined wavelength (in this example, the luminance). Thus, the measurement unit 50 is controlled. More specifically, after step S112, the measurement unit 50 starts fluorescence measurement (step S114). While moving the measurement unit 50 on the slide 52, fluorescence measurement is performed on the plurality of reaction vessels 100. The PCR result can be confirmed by another method such as electrophoresis without performing fluorescence measurement.

  By controlling the measurement unit 50 to measure the intensity of light of a predetermined wavelength after the third treatment, the reaction solution 140 is correlated with the amount of specific DNA during the period in which the reaction solution 140 is held at the annealing and extension temperature. The intensity of light of a predetermined wavelength can be measured. Therefore, the thermal cycle apparatus 1 suitable for real-time PCR can be realized.

  After the third process, the control unit 40 controls the drive mechanism 30 when the placement unit 15, the first heating unit 21, and the second heating unit 22 are arranged in the first arrangement for a fourth time. A fourth process for controlling the drive mechanism 30 so as to switch the arrangement of the mounting unit 15, the first heating unit 21, and the second heating unit 22 from the first arrangement to the second arrangement by outputting the signal S3. And the third process may be repeated a predetermined number of times.

  More specifically, first, after step S114, the control unit 40 determines whether or not the fourth time has elapsed since step S112 was completed (step S116). In this specific example, the fourth time is the time required for annealing and extension in PCR. When the control unit 40 determines that the fourth time has not elapsed (NO in step S116), the control unit 40 repeats step S116. When the control unit 40 determines that the fourth time has elapsed (YES in step S116), the control unit 40 determines whether or not a predetermined number of cycles has been reached (step S118).

  When the control unit 40 determines that the predetermined number of cycles has not been reached (NO in step S118), the control unit 40 outputs a control signal S3 to the drive mechanism 30 to thereby attach the mounting unit 15. The drive mechanism 30 is controlled so as to switch the arrangement of the first heating unit 21 and the second heating unit 22 from the first arrangement to the second arrangement (step S120). After step S120, steps S110 to S118 are repeated. If the control unit 40 determines that the predetermined number of cycles has been reached (YES in step S118), the process ends. In this specific example, the end of the process is determined based on the number of cycles, but before step S118, it is determined whether or not the intensity of the light of the predetermined wavelength measured in step S114 has reached a predetermined value. If a step is added and the light intensity reaches a predetermined value, the process may be terminated without performing step S118.

  In the third process, the reaction liquid 140 is maintained at the third temperature until the third time elapses in the second arrangement, and in the fourth process, the reaction liquid 140 is maintained at the second temperature until the fourth time elapses in the first arrangement. Retained. Thus, by repeating the fourth process and the third process (more specifically, step S120 and step S110 to step S118), a thermal cycle suitable for PCR can be repeated a predetermined number of times.

4). Examples Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the examples.

4-1. First Example In the first example, an example in which an experiment for measuring the time from when the drive mechanism 30 starts to rotate until the reaction solution 140 starts to move will be described.

The average inner diameter of the reaction vessel 100 is 2.3 mm, the length in the longitudinal direction of the reaction vessel 100 is 37 mm, the rotation radius is 18.5 mm, and the liquid 130 is dimethyl silicone oil (main component polydimethylsiloxane, preparation kinematic viscosity 4 μm ² / s. ), The reaction solution 140 is a 1.0 μl RT-PCR reaction solution (reaction solution having the composition shown in FIG. 10, see the second embodiment for the outline). The reaction liquid 140 exists as a droplet in the liquid 130. The first arrangement and the second arrangement are arrangements in which the longitudinal direction of the reaction vessel 100 is the vertical direction, and the first arrangement is an arrangement in which the sealing portion 120 is above in the direction in which gravity acts, The arrangement 2 is an arrangement in which the sealing portion 120 is located below in the direction in which gravity acts. That is, the drive mechanism 30 rotates the mounting unit 15 and the temperature gradient forming unit 20 by 180 ° when switching between the first arrangement and the second arrangement.

  FIG. 8 is a table showing experimental results in the first example. In order from the top, the driving mechanism 30 rotates the mounting portion 15 and the temperature gradient forming unit 20 at a rotational speed [rotation / second], the ratio of centrifugal force acting on the reaction liquid 140 to gravity, and the driving mechanism 30 includes the mounting portion 15 and the temperature. Time (X) [seconds] required for half-rotating the gradient forming unit 20, time (Y) [seconds] from when the driving mechanism 30 finishes rotating until the reaction solution 140 leaves the reaction vessel 100, the driving mechanism 30 Represents the time (X + Y) [seconds] from the start of rotation to the start of movement of the reaction solution 140.

  FIG. 9 is a graph showing experimental results in the first example. The horizontal axis in FIG. 9 is the rotation speed [rotation / second] at which the drive mechanism 30 rotates the mounting unit 15 and the temperature gradient forming unit 20, and the vertical axis is the time from when the drive mechanism 30 starts to rotate until the reaction solution 140 starts to move. Represents time (X + Y) [seconds].

  In the range where the experiment was performed, the reaction solution 140 did not move in the reaction vessel 100 until the drive mechanism 30 finished rotating, and started moving after the drive mechanism 30 finished rotating. Therefore, the time (X) required for the drive mechanism 30 to rotate the mounting portion 15 and the temperature gradient forming unit 20 half-turn and the time (Y) from when the drive mechanism 30 finishes rotating until the reaction solution 140 leaves the reaction vessel 100. ) And the time (X + Y) from when the driving mechanism 30 starts to rotate until the reaction solution 140 starts to move.

  As shown in FIGS. 8 and 9, when the rotational speed at which the drive mechanism 30 rotates the mounting unit 15 and the temperature gradient forming unit 20 is 0.5 [rotation / second] or more, the rotational speed is 0.5 [rotation]. It was found that the time from the start of rotation of the drive mechanism 30 to the start of movement of the reaction solution 140 was shorter than the range below [rotation / second]. The reason is that when the rotation speed is lower than 0.5 [rotation / second], the time required for the drive mechanism 30 to rotate the mounting portion 15 and the temperature gradient forming portion 20 by half is extremely long. .

  As shown in FIGS. 8 and 9, when the rotational speed at which the drive mechanism 30 rotates the mounting unit 15 and the temperature gradient forming unit 20 is 3.67 [rotations / second] or less, the rotational speed is 3.67 [ It has been found that the time from when the drive mechanism 30 starts to rotate until the reaction solution 140 starts to move is shorter than the range exceeding [rotation / second]. The reason is that when the rotational speed is greater than 3.67 [rotations / second], the time from when the drive mechanism 30 finishes rotating until the reaction solution 140 leaves the reaction vessel 100 becomes extremely long. . As the rotational speed increases, the centrifugal force acting on the reaction liquid 140 increases, and it is considered that the phenomenon in which the reaction liquid 140 sticks to the inner wall of the reaction vessel 100 due to the centrifugal force is generated. In the first embodiment, since the radius of rotation is 18.5 mm, the centrifugal force acting on the reaction liquid 140 when the rotation speed is 3.67 [rotation / second] is one times the gravity. is there.

  From the above experimental results, the controller 40 is such that the rotational speed of the drive mechanism 30 is 0.5 rotations / second or more and the magnitude of the centrifugal force acting on the reaction solution 140 is less than or equal to 1 times the gravity. It was found that the time from when the drive mechanism 30 started to rotate until the reaction solution 140 began to move was shortened by controlling the drive mechanism 30 at the same time. Therefore, it turned out that the thermal cycle apparatus 1 which can perform a thermal cycle in a short time is realizable.

  8 and 9, it is more preferable that the rotation speed of the drive mechanism 30 is not less than 0.5 rotations / second and not more than 1.67 rotations / second. If the rotation speed is 0.5 rotations / second or more and 1.67 rotations / second or less, the time from when the drive mechanism 30 starts to rotate until the reaction solution 140 starts to move can be 1.1 seconds or less. Therefore, the thermal cycle can be performed in a shorter time. The rotation speed of the drive mechanism 30 is more preferably 1.00 rotation / second or more and 1.25 rotation / second or less. If the rotational speed is 1.00 rotation / second or more and 1.25 rotation / second or less, the time from when the drive mechanism 30 starts to rotate until the reaction solution 140 starts to move can be 0.55 seconds or less. The heat cycle can be performed in a shorter time.

4-2. 2nd Example In 2nd Example, the example which performed real-time measurement by RT-PCR including a hot start process using the thermal cycle apparatus 1 is demonstrated.

  FIG. 10 is a table showing the composition of the reaction solution 140 in the second embodiment. In FIG. 10, “SuperScript III Platinum” is “SuperScript III Platinum One-Step Quantitative RT-PCR System with ROX” (“Platinum” is a registered trademark, manufactured by Life Technologies) and includes PCR enzyme and reverse transcriptase. ing. As RNA, RNA extracted from a human nasal wipe (human sample) was used. The human sample was subjected to immunochromatography using a commercially available kit (“Espline influenza A & B-N (“ Espline ”is a registered trademark)”, manufactured by Fujirebio Inc.). It was positive. Note that “type A positive” in immunochromatography does not specifically identify the following influenza type A (InfA).

  FIG. 11 shows a forward primer (F primer) and a reverse primer (R primer) corresponding to influenza A (InfA), swine influenza A (SW InfA), swine influenza H1 (SW H1) and ribonuclease P (RNase P). ) And probe (Probe) base sequences. Both are identical to the nucleotide sequences described in “CDC protocol of realtime RTPCR for swine influenza A (H1N1)” (World Health Organization, revised version 30th April 2009). Each of the four types of probes shown in FIG. 11 increases the fluorescence brightness measured with the amplification of the nucleic acid.

The experimental procedure is as shown in the flowchart of FIG. 7. The first temperature is 45 ° C., the second temperature is 58 ° C., the third temperature is 98 ° C., the first time is 60 seconds, the second time is 10 seconds, The third time was 5 seconds, the fourth time was 30 seconds, and the number of cycles of the thermal cycle treatment was 50. In addition, the number of reaction containers 100 attached to the attachment part 15 was four (sample A to sample D).

  Sample A contains a forward primer, a reverse primer and a fluorescent probe corresponding to influenza A (InfA). Sample B contains a forward primer, a reverse primer and a fluorescent probe corresponding to swine influenza A type (SW InfA). Sample C includes a forward primer, a reverse primer and a fluorescent probe corresponding to swine influenza H1 type (SW H1). Sample D includes a forward primer, a reverse primer and a fluorescent probe corresponding to ribonuclease P (RNase P).

  FIG. 12 is a graph showing the relationship between the number of cycles of the thermal cycle process and the measured luminance in the second example. The horizontal axis of FIG. 12 represents the number of cycles of thermal cycle processing, and the vertical axis represents the relative value of luminance.

  As shown in FIG. 12, it can be seen that the brightness of each of the samples A to D is greatly increased from the vicinity where the number of cycles of the thermal cycle process is about 20 to 30 times. This shows that the reverse transcribed cDNA was amplified using RNA as a template. Sample D is an experiment for endogenous control. Since the brightness of Sample D is increased, it was confirmed that DNA (cDNA) derived from a human sample was amplified. Furthermore, since the cDNA was amplified in Sample A to Sample D, it was found that the human sample contained all RNAs of influenza A, swine influenza A, and swine influenza H1. This result is consistent with the result of immunochromatography. Therefore, it was confirmed that real-time measurement can be performed by RT-PCR including a hot start process using the thermal cycling apparatus 1 according to the present embodiment.

  In addition, embodiment mentioned above and a modification are examples, Comprising: It is not necessarily limited to these. For example, a plurality of embodiments and modifications can be combined as appropriate.

  The present invention is not limited to the embodiments and examples described above, and various modifications can be made. For example, the present invention includes configurations that are substantially the same as the configurations described in the embodiments (for example, configurations that have the same functions, methods, and results, or configurations that have the same objects and effects). In addition, the invention includes a configuration in which a non-essential part of the configuration described in the embodiment is replaced. In addition, the present invention includes a configuration that exhibits the same operational effects as the configuration described in the embodiment or a configuration that can achieve the same object. In addition, the invention includes a configuration in which a known technique is added to the configuration described in the embodiment.

DESCRIPTION OF SYMBOLS 1 ... Thermal cycle apparatus, 15 ... Mounting part, 16 ... Fixing member, 17 ... Flange, 18 ... Flange, 19 ... Conductor, 20 ... Temperature gradient formation part, 21 ... 1st heating part, 21a ... 1st heater, 21b ... 1st heat block, 22 ... 2nd heating part, 22a ... 2nd heater, 22b ... 2nd heat block, 30 ... Drive mechanism, 31 ... Bearing, 40 ... Control part, 50 ... Measuring part, 52 ... Slide, 100 ... Reaction vessel, 110 ... channel, 111 ... first region, 112 ... second region, 130 ... liquid, 140 ... reaction solution, 151 ... insertion port, 210 ... fin, S1-S4 ... control signal

SEQ ID NO: 1 is the sequence of the InfA forward primer.
SEQ ID NO: 2 is the sequence of the InfA reverse primer.
SEQ ID NO: 3 is the sequence of the InfA fluorescent probe.
Sequence number 4 is the arrangement | sequence of the forward primer of SW InfA.
SEQ ID NO: 5 is the reverse primer sequence of SW InfA.
Sequence number 6 is the arrangement | sequence of the fluorescent probe of SW InfA.
Sequence number 7 is the arrangement | sequence of the forward primer of SW H1.
Sequence number 8 is the arrangement | sequence of the reverse primer of SW H1.
Sequence number 9 is the arrangement | sequence of the fluorescent probe of SW H1.
SEQ ID NO: 10 is the sequence of RNase P forward primer.
SEQ ID NO: 11 is the sequence of RNase P reverse primer.
SEQ ID NO: 12 is the sequence of a fluorescent probe for RNase P.

Claims (2)

A mounting portion capable of mounting a reaction vessel including a flow path through which the reaction liquid is filled with a liquid having a specific gravity different from that of the reaction liquid and immiscible with the reaction liquid, and the reaction liquid moves;
A temperature gradient forming unit that forms a temperature gradient in a direction in which the reaction solution moves with respect to the flow path when the reaction container is mounted on the mounting unit;
When the mounting portion and the temperature gradient forming portion have a component perpendicular to the direction in which gravity acts, and the reaction vessel is mounted on the mounting portion, the reaction solution moves through the flow path. A drive mechanism that rotates on a rotating shaft having a component perpendicular to the direction;
A control unit for controlling the drive mechanism;
Including
The controller is
Controlling the drive mechanism so that the rotational speed of the drive mechanism is 0.5 revolutions / second or more and the magnitude of the centrifugal force acting on the reaction liquid is 1 or less times the gravity;
The drive mechanism is different from the first arrangement in the arrangement of the mounting part and the temperature gradient forming part in the first arrangement and the position of the lowest point in the flow path in the direction in which the gravity acts. Rotate between the second arrangement,
The drive mechanism includes a case of rotating to the second arrangement from the first arrangement, the case is rotated to the first arrangement from the second arrangement, in the mounting portion and the opposite direction Thermal cycle device that rotates the temperature gradient forming section In claim 1,
The controller is
A process of controlling the drive mechanism so as to maintain the placement of the mounting portion and the temperature gradient forming portion in the second placement;
A process of controlling the drive mechanism so as to switch the arrangement of the mounting part and the temperature gradient forming part from the second arrangement to the first arrangement;
A process of controlling the drive mechanism so as to maintain the placement of the mounting portion and the temperature gradient forming portion in the first placement;
A process of controlling the drive mechanism so as to switch the arrangement of the mounting part and the temperature gradient forming part from the first arrangement to the second arrangement;
And
The controller is
In the process of controlling the drive mechanism to switch from the second arrangement to the first arrangement, and the process of controlling the drive mechanism to switch from the first arrangement to the second arrangement,
A thermal cycle device that controls the drive mechanism so that the rotational speed of the drive mechanism is 0.5 rotations / second or more and the magnitude of the centrifugal force acting on the reaction liquid is 1 time or less of gravity. .