# Patent

## Title
Thermal cycler and control method of thermal cycler

## Abstract

An attachment unit for attachment of a reaction container including a channel filled with a reaction solution containing a fluorescent probe and a liquid having a specific gravity different from that of the reaction solution and being immiscible with the reaction solution, the reaction solution moving close to opposed inner walls, a first heating unit heating a first region of the channel and a second heating unit heating a second region of the channel when the reaction container is attached to the attachment unit, a drive mechanism switching arrangement of the attachment unit, the first heating unit, and the second heating unit between a first arrangement and a second arrangement in which a lowermost position of the channel is located within a first region and a second region, respectively, a measurement unit measuring light intensity, and a control unit controlling the portions described above.

## Background
BACKGROUND
1. Technical Field
The present invention relates to a thermal cycler and a control method of the thermal cycler.
2. Related Art
Recently, with development of utilization technologies of genes, medical treatment utilizing genes such as gene diagnoses and gene therapies has attracted attention, and many techniques using genes for breed identification and breed improvement have been developed in agriculture and livestock fields. As technologies for utilizing genes, a technology such as a PCR (Polymerase Chain Reaction) method has been widespread. Today, the PCR method is an essential technology in elucidation of information of biological materials.
The PCR method is a technique of amplifying target nucleic acid by applying thermal cycling to a solution containing nucleic acid as a target of amplification (target nucleic acid) and reagent (reaction solution). The thermal cycling is processing of periodically applying two or more steps of temperatures to the reaction solution. In the PCR method, generally, thermal cycling of two or three steps is applied.
In the PCR method, generally, a container for biochemical reaction called a tube or a chip for biological sample reaction (biochip) is used. However, in the technique of related art, there have been problems that large amounts of reagent etc. are necessary, equipment becomes complex for realization of thermal cycling necessary for reaction, and the reaction takes time. Accordingly, biochips and reactors for performing PCR with high accuracy in short time using extremely small amounts of reagent and specimen have been required.
In order to solve the problem, Patent Document 1 (JP-A-2009-136250) has disclosed a biological sample reactor of performing thermal cycling by rotating a chip for biological sample reaction filled with a reaction solution and a liquid being immiscible with the reaction liquid and having a lower specific gravity than that of the reaction solution around a rotation axis in the horizontal direction to move the reaction solution.
Further, real time PCR of measuring amplification by PCR over time (in real time) by detecting light having a predetermined wavelength has been known.
The equipment disclosed in Patent Document 1 has applied thermal cycling to a reaction solution by continuously rotating a biochip. However, it has been difficult to hold the reaction solution at a desired temperature in a desired period because the reaction solution moves within a channel of the biochip with the rotation. Accordingly, additional ideas have been required for appropriate real-time PCR.
SUMMARY
An advantage of some aspects of the invention is to provide a thermal cycler and a control method of thermal cycler suitable for real-time PCR.
(1) A thermal cycler according to an aspect of the invention includes an attachment unit for attachment of a reaction container including a channel filled with a reaction solution containing a fluorescent probe that changes intensity of light having a predetermined wavelength by binding to a DNA sequence and a liquid having a specific gravity different from that of the reaction solution and being immiscible with the reaction solution, the reaction solution moving close to opposed inner walls, a first heating unit that heats a first region of the channel when the reaction container is attached to the attachment unit, a second heating unit that heats a second region of the channel different from the first region when the reaction container is attached to the attachment unit, a drive mechanism that switches arrangement of the attachment unit, the first heating unit, and the second heating unit between a first arrangement in which a lowermost position of the channel in a direction in which gravity acts is located within the first region and a second arrangement in which the lowermost position of the channel in the direction in which the gravity acts is located within the second region when the reaction container is attached to the attachment unit, a measurement unit that measures the intensity of the light having the predetermined wavelength, and a control unit that controls the drive mechanism, the first heating unit, the second heating unit, and the measurement unit, wherein the control unit performs first processing of controlling the first heating unit at a first temperature, second processing of controlling the second heating unit at a second temperature higher than the first temperature, third processing of controlling the drive mechanism to switch the arrangement of the attachment unit, the first heating unit, and the second heating unit from the second arrangement to the first arrangement if a first period has elapsed with the arrangement of the attachment unit, the first heating unit, and the second heating unit being the second arrangement, and fourth processing of controlling the measurement unit to measure the intensity of the light having the predetermined wavelength after the third processing.
According to the aspect of the invention, the state in which the reaction container is held in the first arrangement and the state in which the reaction container is held in the second arrangement may be switched by switching the arrangement of the attachment unit, the first heating unit, and the second heating unit. The first arrangement is the arrangement in which the first region of the channel forming the reaction container is located in the lowermost part of the channel in the direction in which the gravity acts. The second arrangement is the arrangement in which the second region of the channel forming the reaction container is located in the lowermost part of the channel in the direction in which the gravity acts. That is, when the specific gravity of the reaction solution is relatively large, the reaction solution may be held in the first region in the first arrangement and the reaction solution may be held in the second region in the second arrangement by the action of the gravity. The first region is heated by the first heating unit and the second region is heated by the second heating unit, and thereby, the first region and the second region may be set at different temperatures. Therefore, the reaction solution may be held at a predetermined temperature while the reaction container is held in the first arrangement or the second arrangement, and the thermal cycler that can easily control the heating period may be provided. Further, the reaction solution is held at the second temperature in the third processing and the reaction solution is held at the first temperature lower than the second temperature in the fourth processing. For example, by setting the first temperature to the annealing and elongation temperature and the second temperature to the denaturation temperature of DNA, PCR may be performed. By controlling the measurement unit to measure the intensity of the light having the predetermined wavelength in the fourth processing, the intensity of the light having the predetermined wavelength emitted by the fluorescent probe binding to the DNA sequence may be measured in the period in which the reaction solution is held at the annealing and elongation temperature. Therefore, the thermal cycler suitable for real-time PCR may be realized.
(2) In the above described thermal cycler, the control unit may further perform fifth processing of allowing a second period to elapse with the arrangement of the attachment unit, the first heating unit, and the second heating unit being the second arrangement after the second processing, and third processing after the fifth processing.
The reaction solution is held at the second temperature in the fifth processing. Generally, in PCR using hot start enzyme, the hot start enzyme is activated at the denaturation temperature (hot start). Therefore, for example, when the second temperature is set to the denaturation temperature, by performing the fifth processing, thermal cycling including hot start may be realized without affecting the first period of the third processing.
(3) In the above described thermal cycler, the control unit may further perform sixth processing of controlling the first heating unit at a third temperature lower than the first temperature and allowing a third period to elapse with the arrangement of the attachment unit, the first heating unit, and the second heating unit being the first arrangement, seventh processing of controlling the drive mechanism to switch the arrangement of the attachment unit, the first heating unit, and the second heating unit from the first arrangement to the second arrangement after the sixth processing, and fifth processing after the seventh processing.
The reaction solution is held at the third temperature lower than the first temperature in the seventh processing. For example, the third temperature may be set to a temperature at which reverse transcription action progresses in RT-PCR (reverse transcription polymerase chain action). Therefore, by performing the seventh processing prior to the fifth processing, the reverse transcription reaction may be performed before PCR, and thus, the thermal cycler suitable for RT-PCR may be realized.
(4) In the above described thermal cycler, the control unit may perform eighth processing of controlling the drive mechanism to switch the arrangement of the attachment unit, the first heating unit, and the second heating unit from the first arrangement to the second arrangement if a fourth period has elapsed with the arrangement of the attachment unit, the first heating unit, and the second heating unit being the first arrangement, the third processing, and the fourth processing repeatedly at a predetermined number of times after the fourth processing.
Thereby, thermal cycling suitable for PCR may be performed repeatedly at a predetermined number of times.
(5) In the above described thermal cycler, the measurement unit may measure intensity of light from a region containing the first region.
Thereby, in the fourth processing, the intensity of the light from the first region in which the reaction solution is held at the annealing and elongation temperature can be measured, and thus, intensity of light having a predetermined wavelength correlated with an amount of specific DNA may be measured more accurately.
(6) A control method of a thermal cycler according to an aspect of the invention is a control method of a thermal cycler, and the thermal cycler includes an attachment unit for attachment of a reaction container including a channel filled with a reaction solution containing a fluorescent probe that changes intensity of light having a predetermined wavelength by binding to a DNA sequence and a liquid having a specific gravity different from that of the reaction solution and being immiscible with the reaction solution, the reaction solution moving close to opposed inner walls, a first heating unit that heats a first region of the channel when the reaction container is attached to the attachment unit, a second heating unit that heats a second region of the channel different from the first region when the reaction container is attached to the attachment unit, a drive mechanism that switches arrangement of the attachment unit, the first heating unit, and the second heating unit between a first arrangement in which a lowermost position of the channel in a direction in which gravity acts is located within the first region and a second arrangement in which the lowermost position of the channel in the direction in which the gravity acts is located within the second region when the reaction container is attached to the attachment unit, and a measurement unit that measures the intensity of the light having the predetermined wavelength, and the control method includes performing first processing of controlling the first heating unit at a first temperature, performing second processing of controlling the second heating unit at a second temperature higher than the first temperature, performing third processing of controlling the drive mechanism to switch the arrangement of the attachment unit, the first heating unit, and the second heating unit from the second arrangement to the first arrangement if a first period has elapsed with the arrangement of the attachment unit, the first heating unit, and the second heating unit being the second arrangement, and performing fourth processing of controlling the measurement unit to measure the intensity of the light having the predetermined wavelength after the third processing.
According to this aspect of the invention, the state in which the reaction container is held in the first arrangement and the state in which the reaction container is held in the second arrangement may be switched by switching the arrangement of the attachment unit, the first heating unit, and the second heating unit. The first arrangement is the arrangement in which the first region of the channel forming the reaction container is located in the lowermost part of the channel in the direction in which the gravity acts. The second arrangement is the arrangement in which the second region of the channel forming the reaction container is located in the lowermost part of the channel in the direction in which the gravity acts. That is, when the specific gravity of the reaction solution is relatively larger, the reaction solution may be held in the first region in the first arrangement and the reaction solution may be held in the second region in the second arrangement by the action of the gravity. The first region is heated by the first heating unit and the second region is heated by the second heating unit, and thereby, the first region and the second region may be set at different temperatures. Therefore, the reaction solution may be held at a predetermined temperature while the reaction container is held in the first arrangement or the second arrangement, and the control method of the thermal cycler that can easily control the heating period may be provided. Further, the reaction solution is held at the second temperature in the third processing and the reaction solution is held at the first temperature lower than the second temperature in the fourth processing. For example, by setting the first temperature to the annealing and elongation temperature and the second temperature to the denaturation temperature, PCR may be performed. By controlling the measurement unit to measure the intensity of the light having the predetermined wavelength in the fourth processing, the intensity of the light having the predetermined wavelength emitted by the fluorescent probe binding to the DNA sequence may be measured in the period in which the reaction solution is held at the annealing and elongation temperature. Therefore, the control method of the thermal cycler suitable for real-time PCR may be realized.



BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be described with reference to the accompanying drawings, wherein like numbers reference like elements.
FIG. 1 is a perspective view of a thermal cycler according to an embodiment.
FIG. 2 is an exploded perspective view of a main body of the thermal cycler according to the embodiment.
FIG. 3 is a vertical sectional view along A-A line in FIG. 1.
FIG. 4 is a sectional view showing a configuration of a reaction container to be attached to the thermal cycler according to the embodiment.
FIG. 5 is a functional block diagram of the thermal cycler according to the embodiment.
FIG. 6A is a sectional view schematically showing a section in a plane passing through the A-A line of FIG. 1A and perpendicular to a rotation axis in a first arrangement, and FIG. 6B is a sectional view schematically showing a section in the plane passing through the A-A line of FIG. 1A and perpendicular to the rotation axis in a second arrangement.
FIG. 7 is a flowchart for explanation of a first specific example of a control method of the thermal cycler according to the embodiment.
FIG. 8 is a flowchart for explanation of a second specific example of a control method of the thermal cycler according to the embodiment.
FIG. 9 is a flowchart for explanation of a third specific example of a control method of the thermal cycler according to the embodiment.
FIG. 10 is a table showing a composition of a reaction solution in a first working example.
FIG. 11 is a table showing base sequences of forward primers (F primers), reverse primers (R primers), and probes.
FIG. 12 is a graph showing relationships between the number of cycles of thermal cycling processing and measured brightness in the first working example.
FIG. 13 is a table showing a composition of the reaction solution in a second working example.
FIG. 14 is a graph showing relationships between the number of cycles of thermal cycling processing and measured brightness in the second working example.



DESCRIPTION OF EXEMPLARY EMBODIMENTS
As below, preferred embodiments of the invention will be explained in detail using the drawings. Note that the embodiments to be explained do not unduly limit the invention described in the appended claims. Further, not all of the configurations to be explained are essential component elements of the invention.
1. Overall Configuration of Thermal Cycler According to Embodiment
FIG. 1 is a perspective view of a thermal cycler 1 according to an embodiment. FIG. 2 is an exploded perspective view of a main body 10 of the thermal cycler 1 according to the embodiment. FIG. 3 is a vertical sectional view along A-A line in FIG. 1. In FIG. 3, arrow g indicates a direction in which gravity acts.
The thermal cycler 1 according to the embodiment includes an attachment unit 15 for attachment of a reaction container 100 including a channel 110 filled with a reaction solution 140 containing a fluorescent probe that changes intensity of light having a predetermined wavelength by binding to a DNA (Deoxyribonucleic acid) sequence and a liquid 130 having a specific gravity different from that of the reaction solution 140 and being immiscible with the reaction solution 140, the reaction solution 140 moving close to opposed inner walls (the details will be described later in section of “2. Configuration of Reaction Container attached to Thermal Cycler according to Embodiment”), a first heating unit 21 that heats a first region 111 of the channel 110 when the reaction container 100 is attached to the attachment unit 15, a second heating unit 22 that heats a second region 112 of the channel 110 different from the first region 111 when the reaction container 100 is attached to the attachment unit 15, a drive mechanism 30 that switches arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 between a first arrangement in which the lowermost position of the channel 110 in a direction in which gravity acts is located within the first region 111 and a second arrangement in which the lowermost position of the channel 110 in the direction in which the gravity acts is located within the second region 112 when the reaction container 100 is attached to the attachment unit 15, a measurement unit 50 that measures the intensity of the light having the predetermined wavelength, and a control unit 40 that controls the drive mechanism 30, the first heating unit 21, the second heating unit 22, and the measurement unit 50.
In the example shown in FIG. 1, the thermal cycler 1 includes the main body 10 and the drive mechanism 30. As shown in FIG. 2, the main body 10 includes the attachment unit 15, the first heating unit 21, and the second heating unit 22.
The attachment unit 15 has a structure to which the reaction container 100 is attached. In the example shown in FIGS. 1 and 2, the attachment unit 15 of the thermal cycler 1 has a slot structure with an insertion opening 151 into which the reaction container 100 is attached by insertion from the insertion opening 151. In the example shown in FIG. 2, the attachment unit 15 has a structure in which the reaction container 100 is inserted into a hole penetrating a first heat block 21b of the first heating unit 21 and a second heat block 22b of the second heating unit 22. The first heat block 21b and the second heat block 22b will be described later. A plurality of the attachment units 15 may be provided in the main body 10, and ten attachment units 15 are provided in the main body 10 in the example shown in FIGS. 1 and 2. Further, in the example shown in FIGS. 2 and 3, the attachment unit 15 is formed as a part of the first heating unit 21 and the second heating unit 22, however, the attachment unit 15 and the first heating unit 21 and the second heating unit 22 may be formed as separate members as long as the positional relationship between them may not change when the drive mechanism 30 is operated.
Note that, in the embodiment, the example in which the attachment unit 15 has the slot structure has been shown, however, the attachment unit 15 has any structure as long as it may hold the reaction container 100. For example, a structure of fitting the reaction container 100 in a recess that conforms to the shape of the reaction container 100 or a structure of sandwiching and holding the reaction container 100 may be employed.
The first heating unit 21 heats the first region 111 of the channel 110 of the reaction container 100 when the reaction container 100 is attached to the attachment unit 15. In the example shown in FIG. 3, the first heating unit 21 is located in a position for heating the first region 111 of the reaction container 100 in the main body 10.
The first heating unit 21 may include a mechanism of generating heat and a member of transmitting the generated heat to the reaction container 100. In the example shown in FIG. 2, the first heating unit 21 includes a first heater 21a as a mechanism of generating heat and the first heat block 21b as a member of transmitting the generated heat to the reaction container 100.
In the thermal cycler 1, the first heater 21a is a cartridge heater and connected to an external power supply (not shown) by a conducting wire 19. The first heater 21a is not limited but includes a carbon heater, a sheet heater, an IH heater (electromagnetic induction heater), a Peltier device, a heating liquid, a heating gas, etc. The first heater 21a is inserted into the first heat block 21b and the first heater 21a generates heat to heat the first heat block 21b. The first heat block 21b is a member of transmitting the heat generated from the first heater 21a to the reaction container 100. In the thermal cycler 1, the first heat block 21b is an aluminum block. The cartridge heater is easily temperature-controlled, and, with the cartridge heater for the first heater 21a, the temperature of the first heating unit 21 may be easily stabilized. Therefore, more accurate thermal cycling may be realized.
The material of the heat block may be appropriately selected in consideration of conditions of coefficient of thermal conductivity, heat retaining characteristics, ease of working, etc. For example, aluminum has a high coefficient of thermal conductivity, and, by forming the first heat block 21b using aluminum, the reaction container 100 may be efficiently heated. Further, unevenness in heating is hard to be produced in the heat block, and the thermal cycling with high accuracy may be realized. Furthermore, working is easy, and the first heat block 21b may be molded with high accuracy and the heating accuracy may be improved. Therefore, more accurate thermal cycling may be realized. Note that, for the material of the heat block, for example, copper alloy may be used or several materials may be combined.
It is preferable that the first heating unit 21 is in contact with the reaction container 100 when the attachment unit 15 is attached to the reaction container 100. Thereby, when the reaction container 100 is heated by the first heating unit 21, the heat of the first heating unit 21 may be transmitted to the reaction container 100 more stably than in the configuration in which the first heating unit 21 is not in contact with the reaction container 100, and thus, the temperature of the reaction container 100 may be stabilized. When the attachment unit 15 is formed as the part of the first heating unit 21 like in the embodiment, it is preferable that the attachment unit 15 is in contact with the reaction container 100. Thereby, the heat of the first heating unit 21 may be stably transmitted to the reaction container 100, and the reaction container 100 may be efficiently heated.
The second heating unit 22 heats the second region 112 of the channel 110 of the reaction container 100 nearer the insertion opening 151 than the first region 111 to a second temperature different from the first temperature when the attachment unit 15 is attached to the reaction container 100. In the example shown in FIG. 3, the second heating unit 22 is located in a position for heating the second region 112 of the reaction container 100 in the main body 10. 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 embodiment is the same as that of the first heating unit 21 except that the region of the reaction container 100 to be heated and the temperature of heating are different from those of the first heating unit 21. Note that different heating mechanisms may be employed in the first heating unit 21 and the second heating unit 22. Further, the materials of the first heat block 21b and the second heat block 22b may be different.
The first heating unit 21 and the second heating unit 22 function as a temperature gradient forming section of forming a temperature gradient in a direction in which the reaction solution 140 moves for the channel 110 when the attachment unit 15 is attached to the reaction container 100. Here, “forming a temperature gradient” refers to forming a state in which a temperature changes along a predetermined direction. Therefore, “forming a temperature gradient in a direction in which the reaction solution 140 moves” refers to forming a state in which a temperature changes in a direction in which the reaction solution 140 moves. “A state in which a temperature changes along a predetermined direction” may refer to a state in which a temperature monotonically becomes higher or lower along a predetermined direction, or a state in which a temperature is changed in the middle from the change to be higher to the change to be lower or from the change to be lower to the change to be higher along a predetermined direction. In the main body 10 of the thermal cycler 1, the first heating unit 21 is located at the side farther from the insertion opening 151 of the attachment unit 15 and the second heating unit 22 is located at the side nearer the insertion opening 151 of the attachment unit 15.
Further, the first heating unit 21 and the second heating unit 22 are provided separately from each other in the main body 10. Thereby, the first heating unit 21 and the second heating unit 22 controlled at the different temperatures from each other are hard to affect each other, and the temperatures of the first heating unit 21 and the second heating unit 22 may be 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 thermal cycler 1, the first heating unit 21 and the second heating unit 22 are fixed on their peripheries by a fixing member 16, a flange 17, and a flange 18. The flange 18 is supported by a bearing 31. Note that the number of heating units may be an arbitrary number equal to or more than two as long as the temperature gradient is formed to a degree that may secure desired reaction accuracy.
The temperatures of the first heating unit 21 and the second heating unit 22 may be controlled by a temperature sensor (not shown) and the control unit 40 to be described later. It is preferable that the temperatures of the first heating unit 21 and the second heating unit 22 are set so that the reaction container 100 may be heated to a desired temperature. The details of the control of the temperatures of the first heating unit 21 and the second heating unit 22 will be described in the section of “3. Control Example of Thermal Cycler”. Note that it is only necessary that the temperatures of the first heating unit 21 and the second heating unit 22 are controlled so that the first region 111 and the second region 112 of the reaction container 100 may be heated to desired temperatures. For example, in consideration of the material and the size of the reaction container 100, the temperatures of the first region 111 and the second region 112 may be heated to the desired temperatures more accurately. In the embodiment, the temperatures of the first heating unit 21 and the second heating unit 22 are measured by a temperature sensor. The temperature sensor of the embodiment is a thermocouple. Note that the temperature sensor is not limited but may include a temperature sensing resistor or a thermistor, for example.
The drive mechanism 30 switches the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 between the first arrangement in which the lowermost position of the channel 110 in the direction in which the gravity acts is located within the first region 111 and the second arrangement in which the lowermost position of the channel 110 in the direction in which the gravity acts is located within the second region 112 when the reaction container 100 is attached to the attachment unit 15. In the embodiment, the drive mechanism 30 is a mechanism of rotating the attachment unit 15, the first heating unit 21, and the second heating unit 22 around the rotation axis R having a component perpendicular to the direction in which the gravity acts and a component perpendicular to the direction in which the reaction solution 140 moves in the channel 110 when the attachment unit 15 is attached to the reaction container 100.
The direction “having a component perpendicular to the direction in which the gravity acts” refers to a direction having a component perpendicular to the direction in which the gravity acts when the direction is expressed by a vector sum of “a component in parallel to the direction in which the gravity acts” and “a component perpendicular to the direction in which the gravity acts”.
The direction “having a component perpendicular to the direction in which the reaction solution 140 moves in the channel 110” refers to a direction having a component perpendicular to the direction in which the reaction solution 140 moves in the channel 110 when the direction is expressed by a vector sum of “a component in parallel to the direction in which the reaction solution 140 moves in the channel 110” and “a component perpendicular to the direction in which the reaction solution 140 moves in the channel 110”.
In the thermal cycler 1 of the embodiment, the drive mechanism 30 rotates the attachment unit 15, the first heating unit 21, and the second heating unit 22 around the same rotation axis R. Further, in the embodiment, the drive mechanism 30 includes a motor and a drive shaft (not shown), and the drive shaft and the flange 17 of the main body 10 are connected. When the motor of the drive mechanism 30 is operated, the main body 10 is rotated around the drive axis as the rotation axis R. In the embodiment, ten attachment unit 15 are provided along the direction of the rotation axis R. Note that, as the drive mechanism 30, not limited to the motor, but, for example, a handle, a spiral spring, or the like may be employed.
The thermal cycler 1 includes the measurement unit 50. The measurement unit 50 measures intensity of light having a predetermined wavelength. In the embodiment, a fluorescence detector is employed as the measurement unit 50. Thereby, the thermal cycler 1 may be used for application with fluorescence measurement such as real-time PCR, for example. The number of measurement units 50 is arbitrary as long as the measurement may be performed without difficulty. In the example shown in FIG. 1, the fluorescence measurement is performed while one measurement unit 50 is moved along a slide 52.
It is more preferable that the measurement unit 50 is located at the side nearer the first heating unit 21 than at the side nearer the second heating unit 22. Thereby, the measurement unit hardly becomes an obstacle to the operation when the attachment unit 15 is attached to the reaction container 100. Further, the measurement unit 50 may be provided to measure light from a region containing the first region 111 of the reaction container 100. When the temperature of the first heating unit 21 is set to an annealing and elongation temperature (a temperature at which annealing and elongation reaction progresses) of PCR, the intensity of the light having the predetermined wavelength correlated with an amount of specific DNA may be measured more accurately. Therefore, appropriate fluorescence measurement may be performed in real-time PCR. Furthermore, when a reaction container 100 with a lid (sealing part 120) to be described later is used, more appropriate fluorescence measurement may be performed in the first region 111 at the side farther from the lid than in the second region 112 at the side nearer the lid because there are less members between the measurement unit 50 and the reaction solution 140.
As described above, when the thermal cycler 1 is used for real-time PCR, in a period in which thermal cycling necessary for PCR is applied to the reaction solution 140, it is preferable that the measurement unit 50 is provided at the side nearer the first heating unit 21 and the first heating unit 21 is set to the annealing and elongation temperature of PCR (about 50° C. to 75° C.). In this case, the second heating unit 22 nearer the insertion opening 151 is set to a thermal denaturation temperature (about 90° C. to 100° C.) higher than the annealing and elongation temperature of PCR.
The thermal cycler 1 includes the control unit 40. The control unit 40 controls the first heating unit 21, the second heating unit 22, the drive mechanism 30, and the measurement unit 50. A control example by the control unit 40 will be described in detail in the section of “3. Control Example of Thermal Cycler”. The control unit 40 may be adapted to be realized by a dedicated circuit and perform the control to be described later. Further, the control unit 40 may be adapted to function as a computer using a CPU (Central Processing Unit), for example, by executing control programs stored in a memory device such as a ROM (Read Only Memory) or a RAM (Random Access Memory) and perform the control to be described later. In this case, the memory device may have a work area that temporarily stores intermediate data and control results with the control. Further, the control unit 40 may have a timer for measuring time. Furthermore, the control unit 40 may control the first heating unit 21 and the second heating unit 22 to desired temperatures based on the output of the above described temperature sensor (not shown).
It is preferable that the thermal cycler 1 includes a structure of holding the reaction container 100 in a predetermined position with respect to the first heating unit 21 and the second heating unit 22. Thereby, a predetermined regions of the reaction container 100 may be heated by the first heating unit 21 and the second heating unit 22. More specifically, the first region 111 and the second region 112 of the channel 110 forming the reaction container 100 may be heated by the first heating unit 21 and the second heating unit 22, respectively. In the embodiment, by appropriately setting the sizes of through holes provided in the first heat block 21b and the second heat block 22b (the diameter of the attachment unit 15), the reaction container 100 may be held in a predetermined position with respect to the first heating unit 21 and the second heating unit 22.
The first heat block 21b may have a structure with fins 210. Thereby, the surface area of the first heating unit becomes larger and the time taken for changing the temperature of the first heating unit 21 from the higher temperature to the lower temperature becomes shorter.
The thermal cycler 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 heat transfer between the first heating unit 21 and the second heating unit 22 may be suppressed. Therefore, the first heating unit 21 and the second heating unit 22 controlled at the different temperatures from each other become harder to affect each other, and thus, the temperatures of the first heating unit 21 and the second heating unit 22 may be easily stabilized.
2. Configuration of Reaction Container Attached to Thermal Cycler According to Embodiment
FIG. 4 is a sectional view showing a configuration of the reaction container 100 attached to thermal cycler 1 according to the embodiment. In FIG. 4, arrow g indicates a direction in which gravity acts.
The reaction container 100 includes the channel 110 filled with the reaction solution 140 containing a fluorescent probe that changes intensity of light having a predetermined wavelength by binding to a DNA sequence and a liquid 130 having a different specific gravity from that of the reaction solution 140 and being immiscible with the reaction solution 140 (hereinafter, referred to as “liquid 130”), in which the reaction solution 140 moves along the opposed inner walls. In the embodiment, the liquid 130 is a liquid having a lower specific gravity than that of the reaction solution 140 and being immiscible with the reaction solution 140. Note that, as the liquid 130, for example, a liquid being immiscible with the reaction solution 140 and having a higher specific gravity than that of the reaction solution 140 may be employed. In the example shown in FIG. 4, the reaction container 100 includes the channel 110 and the sealing part 120. The channel 110 is filled with the reaction solution 140 and the liquid 130, and sealed by the sealing part 120.
Note that another dye for real-time PCR than the fluorescent probe may be used. For example, an intercalator having fluorescence that changes by non-specifically binding to double-stranded DNA (by binding regardless of the sequence of DNA) may be used. Examples of the intercalator include SYBR Green (SYBR is a registered trademark) etc. The fluorescence intensity correlates with the amount of amplified DNA, and thus, by the measurement of the fluorescence intensity, whether or not the DNA has been amplified may be determined or the amount of amplified DNA may be estimated. Further, “predetermined wavelength” refers to a wavelength or wavelength band of light emitted by the dye for real-time PCR at which the intensity changes when the binding state of the dye for real-time PCR and the DNA changes.
The channel 110 is formed so that the reaction solution 140 may move along the opposed inner walls. Here, “opposed inner walls” of the channel 110 refer to two regions having an opposed positional relationship on the wall surfaces of the channel 110. “Along” refers to a state in which a distance from the reaction solution 140 to 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 opposed inner walls” refers to “the reaction solution 140 moves in a state in which the distances from the wall surface of the channel 110 to both two regions in the opposed positional relationship are short”. In other words, the distance between the opposed two inner walls of the channel 110 is a distance to a degree that the reaction solution 140 moves along the inner walls.
When the channel 110 of the reaction container 100 has the above described shape, the direction in which the reaction solution 140 moves within the channel 110 may be regulated, and thus, the path in which the reaction solution 140 moves within the channel 110 may be defined to some degree. Thereby, the time taken for the reaction solution 140 to move within the channel 110 may be restricted within a certain range. Therefore, it is preferable that the distance between the opposed two inner walls of the channel 110 is a distance to a degree at which variations in thermal cycling conditions applied to the reaction solution 140 produced by variations in time for the reaction solution 140 to move within the channel 110 may satisfy desired accuracy, i.e., a degree at which the reaction result may satisfy desired accuracy. More specifically, it is desirable that the distance in the direction perpendicular to the direction in which the reaction solution 140 between the opposed two inner walls of the channel 110 moves is a distance to a degree not exceeding two or more droplets of the reaction solution 140.
In the example shown in FIG. 4, the outer shape of the reaction container 100 is a circular truncated cone shape, and the channel 110 in the direction along the center axis (the vertical direction in FIG. 4) as the longitudinal direction is formed. The shape of the channel 110 is a circular truncated cone shape with a section in the direction perpendicular to the longitudinal direction of the channel 110, i.e., a section perpendicular to the direction in which the reaction solution 140 moves in a certain region of the channel 110 (this refers to “section” of the channel 110) in a circular shape. Therefore, in the reaction container 100, the opposed inner walls of the channel 110 are regions containing two points on the wall surface of the channel 110 opposed with the center of the section of the channel 110 in between. Further, “the direction in which the reaction solution 140 moves” is the longitudinal direction of the channel 110.
Note that the shape of the channel 110 is not limited to the truncated cone shape, but may be a columnar shape, for example. Further, the section shape of the channel 110 is not limited to the circular shape, but may be any of a polygonal shape or an oval shape as long as the reaction solution 140 may move along the opposed inner walls. For example, when the section of the channel 110 of the reaction container 100 has a polygonal shape, if a channel having a circular section inscribed in the channel 110 is assumed, “opposed inner walls” are opposed inner walls of the channel. That is, it is only necessary that the channel 110 is formed so that the reaction solution 140 may move along opposed inner walls of a virtual channel having a circular section inscribed in the channel 110. Thereby, even when the section of the channel 110 has a polygonal shape, a path in which the reaction solution 140 moves between the first region 111 and the second region 112 may be defined to some degree. Therefore, the time taken for the reaction solution 140 to move between the first region 111 and the second region 112 may be restricted within a certain range.
The first region 111 of the reaction container 100 is a partial region of the channel 110 to be heated by the first heating unit 21. The second region 112 is a partial region of the channel 110 different from the first region 111 to be heated by the second heating unit 22. In the example shown in FIG. 4, the first region 111 is a region containing one end part in the longitudinal direction of the channel 110, and the second region 112 is a region containing the other end part in the longitudinal direction of the channel 110. In the example shown in FIG. 4, the region surrounded by a dotted line containing the end part at the side farther from the sealing part 120 of the channel 110 is the first region 111, and the region surrounded by a dotted line containing the end part at the side nearer the sealing part 120 of the channel 110 is the second region 112. In the thermal cycler 1 according to the embodiment, the first heating unit 21 heats the first region 111 of the reaction container 100 and the second heating unit 22 heats the second region 112 of the reaction container 100, and thereby, a temperature gradient is formed in the direction in which the reaction solution 140 moves with respect to the channel 110 of the reaction container 100.
The channel 110 is filled with the liquid 130 and the reaction solution 140. The liquid 130 has a property of being immiscible, i.e., unmixed with the reaction solution 140, and the reaction solution 140 is held in droplets in the liquid 130 as shown in FIG. 4. The reaction solution 140 has the higher specific gravity than that of the liquid 130 and is located in the lowermost region of the channel 110 in the direction in which the gravity acts. As the liquid 130, for example, dimethyl silicone oil or paraffin oil may be used. The reaction solution 140 is a liquid containing components necessary for reaction. For example, when the reaction is real-time PCR, the reaction solution 140 contains DNA as a target (to be amplified), DNA polymerase necessary for amplification of the DNA, primer, etc. in addition to the fluorescent probe. When the reaction is RT-PCR, the reaction solution further contains reverse transcriptase enzyme, RNA as a template of reverse transcription, and reverse-transcribed cDNA. For example, when PCR is performed using an oil as the liquid 130, it is preferable that the reaction solution 140 is a solution containing the above described components.
3. Control Example of Thermal Cycler
FIG. 5 is a functional block diagram of the thermal cycler 1 according to the embodiment. The control unit 40 controls the temperature of the first heating unit 21 by outputting a control signal S1 to the first heating unit 21. The control unit 40 controls the temperature of the second heating unit 22 by outputting a control signal S2 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 thermal cycler 1 according to the embodiment will be explained. As below, control of rotating the attachment unit 15, the first heating unit 21, and the second heating unit 22 between the first arrangement in which the lowermost position of the channel 110 in the direction in which the gravity acts is located within the first region 111 and the second arrangement in which the lowermost position of the channel 110 in the direction in which the gravity acts is located within the second region 112 when the reaction container 100 is attached to the attachment unit 15 will be explained as an example.
FIG. 6A is a sectional view schematically showing a section in a plane passing through the A-A line of FIG. 1A and perpendicular to a rotation axis R in the first arrangement, and FIG. 6B is a sectional view schematically showing a section in the plane passing through the A-A line of FIG. 1A and perpendicular to the rotation axis R in the second arrangement. In FIGS. 6A and 6B, white arrows indicate rotation directions of the main body 10 and arrows g indicate the direction in which the gravity acts.
As shown in FIG. 6A, the first arrangement is an arrangement in which, when the attachment unit 15 is attached to the reaction container 100, the first region 111 is located in the lowermost part of the channel 110 in the direction in which the gravity acts. In the example shown in FIG. 6A, in the first arrangement, the reaction solution 140 having the higher specific gravity than that of the liquid 130 exists in the first region 111. Further, as shown in FIG. 6B, the second arrangement is an arrangement in which, when the attachment unit 15 is attached to the reaction container 100, the second region 112 is located in the lowermost part of the channel 110 in the direction in which the gravity acts. In the example shown in FIG. 6B, in the second arrangement, the reaction solution 140 having the higher specific gravity than that of the liquid 130 exists in the second region 112.
In this manner, the drive mechanism 30 rotates the attachment 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, and thereby, thermal cycling may be applied to the reaction solution 140.
According to the embodiment, by switching the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22, the state in which the reaction container 100 is held in the first arrangement and the state in which the reaction container 100 is held in the second arrangement may be switched. The first arrangement is the arrangement in which the first region 111 of the channel 110 forming the reaction container 100 is located in the lowermost part of the channel 110 in a direction in which the gravity acts. The second arrangement is the arrangement in which the second region 112 of the channel 110 forming the reaction container 100 is located in the lowermost part of the channel 110 in the direction in which the gravity acts. That is, when the specific gravity of the reaction solution 140 is larger than that of the liquid 130, the reaction solution 140 may be held in the first region 111 in the first arrangement and the reaction solution 140 may be held in the second region 112 in the second arrangement by the action of the gravity. 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, and thereby, the first region 111 and the second region 112 may be set at different temperatures. Therefore, while the reaction container 100 is held in the first arrangement or the second arrangement, the reaction solution 140 may be held at a predetermined temperature, and thus, the thermal cycler 1 that can easily control the heating period may be provided.
The drive mechanism 30 may rotate the attachment unit 15, the first heating unit 21, and the second heating unit 22 in opposite directions when rotating them from the first arrangement to the second arrangement and when rotating them from the second arrangement to the first arrangement. Thereby, a special mechanism for reducing twisting of wires such as the conducting wire 19 caused by rotation is unnecessary. Therefore, thermal cycler 1 suitable for downsizing may be realized. Further, it is preferable that the number of rotations for rotation from the first arrangement to the second arrangement and the number of rotations for rotation from the second arrangement to the first arrangement are less than one (the rotation angle is less than 360°). Thereby, the degree of twisting of the wires may be reduced. Alternately, as shown in FIGS. 1 and 2, the configuration in which the flange 18 can take up the conducting wire 19 may be employed.
3-1. First Specific Example of Control Method of Thermal Cycler
Next, a first specific example of a control method of the thermal cycler 1 will be explained by taking real-time measurement in two-step temperature PCR as an example. FIG. 7 is a flowchart for explanation of the first specific example of the control method of the thermal cycler 1 according to the embodiment.
In FIG. 7, first, the control unit 40 controls the temperature of the first heating unit 21 at a first temperature (first processing), and controls the temperature of the second heating unit 22 at a second temperature higher than the first temperature (second processing) (step S100). In the specific example, the first temperature is the annealing and elongation temperature in PCR. “Annealing and elongation temperature in PCR” refers to a temperature depending on the type of enzyme for amplification of nucleic acid, and generally within a range from 50° C. to 70° C. In the specific example, the second temperature is the thermal denaturation temperature in PCR. “Thermal denaturation temperature in PCR” is a temperature depending on the type of enzyme for amplification of nucleic acid, and generally within a range from 90° C. to 100° C.
After step S100, the control unit 40 controls the drive mechanism 30 to switch the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 from the first arrangement to the second arrangement (step S102). In thermal cycler 1 shown in FIG. 1, immediately after the reaction container 100 is attached to the attachment unit 15, the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 is the first arrangement and, by performing step S102, the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 is switched to the second arrangement.
Note that the reaction container 100 may be attached to the attachment unit 15 after step S100 and before step S102. Further, in the case of the configuration in which the attachment of the reaction container 100 to the attachment unit 15 is performed when the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 is the second arrangement, step S102 may be unnecessary. When the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 is the second arrangement, the reaction solution 140 is held in the second region 112. That is, the reaction solution 140 is held at the second temperature.
After step S102, the control unit 40 performs third processing of controlling the drive mechanism 30 to switch the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 from the second arrangement to the first arrangement if a first period has elapsed with the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 being the second arrangement.
More specifically, first, the control unit 40 determines whether or not the first period has elapsed after step S102 is ended (step S104). In the specific example, the first period is a period necessary for thermal denaturation in PCR. If the control unit 40 determines that the first period has not elapsed (if NO at step S104), the control unit 40 repeats step S104. If the control unit 40 determines that the first period has elapsed (if YES at step S104), the control unit controls the drive mechanism 30 to switch the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 from the second arrangement to the first arrangement (step S106). When the arrangement of the attachment unit 15, 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, the reaction solution 140 is held at the first temperature. Note that it is only necessary that the first heating unit 21 is at the first temperature in the third processing. That is, the third processing may be performed before the second processing or at the same time with the second processing as long as it is performed after the first processing.
After the third processing, the control unit 40 performs fourth processing of controlling the measurement unit to measure the intensity of the light having the predetermined wavelength. More specifically, after step S106, the measurement unit 50 starts fluorescence measurement (step S108). The fluorescence measurement with respect to plural reaction containers 100 may be performed by moving the measurement unit 50 on the slide 52.
By controlling the measurement unit 50 to measure the intensity of the light having the predetermined wavelength in the fourth processing, the intensity of the light having the predetermined wavelength emitted by the fluorescent probe binding to the DNA sequence may be measured in the period in which the reaction solution 140 is held at the annealing and elongation temperature. Therefore, the thermal cycler 1 suitable for real-time PCR may be realized.
After the fourth processing, the control unit 40 may perform eighth processing of controlling the drive mechanism 30 to switch the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 from the first arrangement to the second arrangement if a fourth period has elapsed with the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 being the first arrangement, the third processing, and the forth processing repeatedly at a predetermined number of times.
More specifically, first, after step S108, the control unit 40 determines whether or not the fourth period has elapsed after step S106 is ended (step S110). In the specific example, the fourth period is a period necessary for annealing and elongation in PCR. If the control unit 40 determines that the fourth period has not elapsed (if NO at step S110), the control unit 40 repeats step S110. If the control unit 40 determines that the fourth period has elapsed (if YES at step S110), the control unit 40 determines whether or not a predetermined number of cycles has been reached (step S112).
If the control unit 40 determines that the predetermined number of cycles has not been reached (if NO at step S112), the control unit 40 controls the drive mechanism 30 to switch the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 from the first arrangement to the second arrangement (step S114). After step S114, steps S104 to S112 are repeated. If the control unit 40 determines that the predetermined number of cycles has been reached (if YES at step S112), the processing is ended.
The reaction solution 140 is held at the second temperature until the first period has elapsed in the second arrangement in the third processing and the fourth processing, and the reaction solution 140 is held at the first temperature until the fourth period has elapsed in the first arrangement in the eighth processing. In this manner, by repeating the eighth processing, the third processing, and the fourth processing (more specifically, step S114 and steps S104 to S112), thermal cycling suitable for PCR may be performed repeatedly at a predetermined number of times.
3-2. Second Specific Example of Control Method of Thermal Cycler
Next, a second specific example of the control method of the thermal cycler 1 will be explained by taking real-time measurement in two-step temperature PCR including a hot start step as an example. FIG. 8 is a flowchart for explanation of the second specific example of the control method of the thermal cycler 1 according to the embodiment. Note that the same steps as those in the first specific example of the control method of thermal cycler 1 shown in FIG. 7 have the same signs, and their detailed explanation will be omitted.
In the second specific example of the control method of the thermal cycler 1, the control unit 40 performs fifth processing of allowing a second period to elapse with the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 being the second arrangement after the second processing, and performs the third processing after the fifth processing.
More specifically, after step S102, the control unit 40 determines whether or not the second period has elapsed after step S102 is ended (step S200). In the specific example, the second period is a period necessary for activation of PCR enzyme. If the control unit 40 determines that the second period has not elapsed (if NO at step S200), the control unit 40 repeats step S200. If the control unit 40 determines that the second period has elapsed (if YES at step S200), the control unit 40 performs step S104. In the specific example, at step S104, the control unit 40 determines whether or not the first period has elapsed after step S200 is ended. Step S106 and the subsequent steps are the same as those of the first specific example of the control method of thermal cycler 1 shown in FIG. 7. Note that it is only necessary that the second heating unit 22 is at the second temperature in the fifth processing. That is, the fifth processing may be performed before the first processing or at the same time with the first processing as long as it is performed after the second processing.
In the example shown in FIG. 8, the reaction solution 140 is held at the second temperature in the fifth processing. In the embodiment, the second processing is the annealing and elongation temperature in PCR and the activation temperature of PCR enzyme. “Activation temperature of PCR enzyme” depends on the type of PCR enzyme, and generally, nearly equal to the annealing and elongation temperature in PCR.
As described above, by performing the fifth processing, thermal cycling including hot start of PCR as a step of activating the PCR enzyme may be realized without affecting the first period of the third processing.
Further, like the first specific example of the control method of the thermal cycler 1 shown in FIG. 7, by controlling the measurement unit 50 to measure the intensity of the light having the predetermined wavelength in the fourth processing, the intensity of the light having the predetermined wavelength emitted by the fluorescent probe binding to the DNA sequence may be measured in the period in which the reaction solution 140 is held at the annealing and elongation temperature. Therefore, the thermal cycler 1 suitable for real-time PCR may be realized.
Furthermore, like the first specific example of the control method of the thermal cycler 1 shown in FIG. 7, by repeating the eighth processing, the third processing, and the fourth processing (more specifically, step S114 and steps S104 to S112), thermal cycling suitable for PCR may be performed repeatedly at a predetermined number of times.
3-3. Third Specific Example of Control Method of Thermal Cycler
Next, a third specific example of the control method of the thermal cycler 1 will be explained by taking real-time measurement in RT-PCR including a hot start step as an example. FIG. 9 is a flowchart for explanation of the third specific example of the control method of the thermal cycler 1 according to the embodiment. Note that the same steps as those in the first specific example of the control method of the thermal cycler 1 shown in FIG. 7 and the second specific example of the control method of the thermal cycler 1 shown in FIG. 8 have the same signs, and their detailed explanation will be omitted.
In the third specific example of the control method of the thermal cycler 1, the control unit 40 performs sixth processing of controlling the first heating unit 21 at a third temperature lower than the first temperature and allowing a third period to elapse with the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 being the first arrangement, performs seventh processing of controlling the drive mechanism 30 to switch the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 from the first arrangement to the second arrangement after the sixth processing, and performs the fifth processing after the seventh processing.
More specifically, first, the control unit 40 controls the temperature of the first heating unit 21 at the third temperature (step S300). In the specific example, the third temperature is a temperature at which reverse transcription action progresses by the reverse transcriptase enzyme. “The temperature at which the reverse transcription action progresses by the reverse transcriptase enzyme” is a temperature depending on the type of the reverse transcriptase enzyme and generally within a range from 20° C. to 70° C., and the more preferable temperature is generally within a range from 40° C. to 50° C. Further, in the specific example, the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 is the first arrangement at the initial operation. Therefore, the reaction solution 140 is held in the first region 111. That is, the reaction solution 140 is held at the third temperature.
Note that, at step S300, the control unit 40 may control the second heating unit 22 at a temperature at which the reverse transcriptase enzyme is not deactivated. “The temperature at which the reverse transcriptase enzyme is not deactivated” is a temperature depending on the type of the reverse transcriptase enzyme, and generally within a range from 20° C. to 70° C. Further, generally, at a temperature exceeding 70° C., the reverse transcriptase enzyme is easily deactivated and deteriorated. Note that “the enzyme is deactivated” refers to that enzyme activity is reduced or lost and the enzyme does not exhibit its own activity even when the experimental condition is adjusted. In this specification, it refers to a state in which the activity of the reverse transcriptase enzyme contained in the reaction solution 140 measured at the optimum temperature of the reverse transcriptase enzyme has been lower than the activity expected for the reverse transcriptase enzyme in the environment (the condition of pH or the like) of the reaction solution. “The temperature at which the reverse transcriptase enzyme is not deactivated” includes the case where the reverse transcriptase enzyme exhibits activity of 100% of the expected enzyme activity and the case where the activity is lower to a degree acceptable in RT-PCR (the case where part of the contained reverse transcriptase enzyme is deactivated). By controlling the temperature of the second heating unit 22 at the temperature at which the reverse transcriptase enzyme is not deactivated, when the reaction container 100 is attached to the attachment unit 15, the reaction solution 140 is not subjected to a high temperature at which the reverse transcriptase enzyme is deactivated.
After step S300, the control unit 40 determines whether or not a third period has elapsed after step S300 is ended (step S302). In the specific example, the third period is a period necessary for reverse transcription reaction. If the control unit 40 determines that the third period has not elapsed (if NO at step S302), the control unit 40 repeats step S302. If the control unit 40 determines that the third period has elapsed (if YES at step S302), the control unit 40 controls the temperature of the first heating unit 21 at the first temperature and controls the temperature of the second heating unit 22 at the second temperature (step S304). The first temperature and the second temperature are the same as those in the first specific example of the control method of the thermal cycler 1 explained using Fit. 7.
After step S304, the control unit 40 controls the drive mechanism 30 to switch the arrangement of the attachment unit 15, the first heating unit 21, and the second heating unit 22 from the first arrangement and the second arrangement (step S306). Therefore, the reaction solution 140 is held in the second region 112. That is, the reaction solution 140 is held at the second temperature.
After step S306, the control unit 40 performs step S200, and the subsequent process is the same as that of the second specific example of the control method of thermal cycler 1 explained using FIG. 8.
In this manner, by performing the seventh processing prior to the fifth processing, the reverse transcription reaction may be performed before PCR, and thus, the thermal cycler 1 suitable for RT-PCR may be realized.
Further, like the first specific example of the control method of the thermal cycler 1 shown in FIG. 7, by controlling the measurement unit 50 to measure the intensity of the light having the predetermined wavelength in the fourth processing, the intensity of the light having the predetermined wavelength emitted by the fluorescent probe binding to the DNA sequence may be measured in the period in which the reaction solution 140 is held at the annealing and elongation temperature. Therefore, the thermal cycler 1 suitable for real-time PCR may be realized.
Furthermore, like the second specific example of the control method of the thermal cycler 1 shown in FIG. 8, by performing the fifth processing, thermal cycling including hot start of PCR as a step of activating the PCR enzyme may be realized without affecting the first period of the third processing.
In addition, like the first specific example of the control method of the thermal cycler 1 shown in FIG. 7, by repeating the eighth processing, the third processing, and the fourth processing (more specifically, step S114 and steps S104 to S112), thermal cycling suitable for PCR may be performed repeatedly at a predetermined number of times.
4. Working Examples
As below, the invention will be more specifically explained using working examples, however, the invention is not limited to the working examples.
4-1. First Working Example
In the first working example, an example of performing two-step temperature real-time PCR using the thermal cycler 1 will be explained.
FIG. 10 is a table showing a composition of the reaction solution 140 in the first working example. In FIG. 10, “SuperScript III Platinum” refers to “SuperScript III Platinum One-Step Quantitative RT-PCR System with ROX (“Platinum” is a registered trademark, (manufactured by Life Technologies))”, and contains PCR enzyme. Regarding the plasmid, samples having known copy numbers were produced by subcloning of PCR reaction products obtained using the primers shown in FIG. 11 in advance. 105 plasmids were added for Sample A, 104 plasmids were added for Sample B, 103 plasmids were added for Sample C, and 102 plasmids were added for Sample D.
FIG. 11 is a table showing base sequences of forward primers (F primers), reverse primers (R primers), and probes corresponding to influenza A virus (InfA), swine influenza A virus (SW InfA), and swine influenza H1 virus (SW H1), ribonuclease P (RNase P). All of them are the same as base sequences described in “CDC protocol of realtime RTPCR for swine influenza A (H1N1)” (World Health Organization, Revised First Edition, Apr. 30, 2009). In all of the four types of probes shown in FIG. 11, fluorescent brightness to be measured increases with amplification of nucleic acid.
The experimental procedure was as shown in the flowcharts in FIG. 8, and the first temperature was 58° C., the second temperature was 98° C., the first period was five seconds, the second period was ten seconds, the fourth period was 30 seconds, and the number of cycles of the thermal cycling processing was 50. Further, the number of reaction containers 100 attached to the attachment unit 15 was four (Sample A to Sample D).
FIG. 12 is a graph showing relationships between the number of cycles of thermal cycling processing and measured brightness in the first working example. The horizontal axis of FIG. 12 indicates the number of cycles of the thermal cycling processing and the vertical axis indicates the relative value of brightness.
As shown in FIG. 12, it is known that, regarding all of Sample A to Sample D, the brightness significantly rose as the number of cycles of the thermal cycling processing was about 20 to 35. Thereby, it is confirmed that DNA has been amplified. Further, from FIG. 12, it is confirmed that the brightness rises more significantly at the less number of cycles in the samples having the larger copy numbers of plasmid, and the number of cycles at which the brightness rises is larger as the concentration of the plasmid contained in the reaction solution 140 is higher.
As described above, it is confirmed that two-step temperature real-time PCR may be performed using the thermal cycler 1 according to the embodiment.
4-2. Second Working Example
In the second working example, an example of performing RT-PCR using the thermal cycler 1 will be explained.
FIG. 13 is a table showing a composition of the reaction solution 140 in the second working example. In FIG. 13, “SuperScript III Platinum” refers to “SuperScript III Platinum One-Step Quantitative RT-PCR System with ROX (“Platinum” is a registered trademark, (manufactured by Life Technologies))”, and contains PCR enzyme and reverse transcriptase enzyme. As RNA, RNA extracted from a human nasal cavity swab (human sample) was used. Note that, regarding the human sample, immuno chromatography was performed using a commercially available kit (“ESPLINE Influenza A&B-N) (ESPLINE is a registered trademark)”, manufactured by FUJIREBIO), and the sample was positive for influenza A virus. Note that “A virus positive” in immuno chromatography does not specifically determine the influenza A virus (InfA). The base sequences of the forward primers (F primers), reverse primers (R primers), probes (Probes) in FIG. 13 are the same as the base sequences shown in FIG. 11.
The experimental procedure was as shown in the flowcharts in FIG. 9, and the first temperature was 58° C., the second temperature was 98° C., the third temperature was 45° C., the first period was five seconds, the second period was ten seconds, the third period was 60 seconds, the fourth period was 30 seconds, and the number of cycles of the thermal cycling processing was 50. Further, the number of reaction containers 100 attached to the attachment unit 15 was four (Sample E to Sample H).
Sample E contains a forward primer, a reverse primer, and a fluorescent probe corresponding to influenza A virus. Sample F contains a forward primer, a reverse primer, and a fluorescent probe corresponding to swine influenza A virus (SW InfA). Sample G contains a forward primer, a reverse primer, and a fluorescent probe corresponding to swine influenza H1 virus (SW H1). Sample H contains a forward primer, a reverse primer, and a fluorescent probe corresponding to ribonuclease P (RNase P).
FIG. 14 is a graph showing relationships between the number of cycles of thermal cycling processing and measured brightness in the second working example. The horizontal axis of FIG. 14 indicates the number of cycles of the thermal cycling processing and the vertical axis indicates the relative value of brightness.
As shown in FIG. 14, it is known that, regarding all of Sample E to Sample H, the brightness significantly rose as the number of cycles of the thermal cycling processing was about 20 to 30. Thereby, it is known that reverse-transcribed cDNA with RNA as the template has been amplified. Sample H was for an experiment of endogenous control, and it is confirmed that DNA (cDNA) derived from the human sample has been amplified because the brightness rose in Sample H. Further, it is known that all RNAs of InfA, SW InfA, SW H1 have been contained in the human sample because cDNA has been amplified in Sample E to Sample H. The result agrees with the result of immuno chromatography. Therefore, it has been confirmed that 1 step RT-PCR may be performed using the thermal cycler 1 according to the embodiment.
Note that the above described embodiment and working example are just examples, and not limited to those. For example, some of the respective embodiments and the respective examples may be appropriately combined.
The invention is not limited to the above described embodiment and example, but other various modifications may be made. For example, the invention includes substantially the same configuration as the configuration explained in the embodiment (for example, a configuration having the same function, method, and result, or a configuration having the same purpose and advantage). Further, the invention includes a configuration in which an insubstantial part of the configuration explained in the embodiment is replaced. Furthermore, the invention includes a configuration that exerts the same effect or a configuration that may achieve the same purpose as that of the configuration explained in the embodiment. In addition, the invention includes a configuration formed by adding a known technology to the configuration explained in the embodiment.
The entire disclosure of Japanese Patent Application No. 2012-079765, filed Mar. 30, 2012 is expressly incorporated by reference herein.
SEQ ID NO: 1 refers to the sequence of the forward primer of InfA.
SEQ ID NO: 2 refers to the sequence of the reverse primer of InfA.
SEQ ID NO: 3 refers to the sequence of the fluorescent probe of InfA.
SEQ ID NO: 4 refers to the sequence of the forward primer of SW InfA.
SEQ ID NO: 5 refers to the sequence of the reverse primer of SW InfA.
SEQ ID NO: 6 refers to the sequence of the fluorescent probe of SW InfA.
SEQ ID NO: 7 refers to the sequence of the forward primer of SW H1.
SEQ ID NO: 8 refers to the sequence of the reverse primer of SW H1.
SEQ ID NO: 9 refers to the sequence of the fluorescent probe of SW H1.
SEQ ID NO: 10 refers to the sequence of the forward primer of RNase P.
SEQ ID NO: 11 refers to the sequence of the reverse primer of RNase P.
SEQ ID NO: 12 refers to the sequence of the fluorescent probe of RNase P.

## Claims

1. A thermal cycler comprising:
an attachment unit for attachment of a removable reaction container, the reaction container including a channel, the channel being filled with a reaction solution containing a fluorescent probe that changes intensity of light having a predetermined wavelength by binding to a DNA sequence and a liquid having a specific gravity different from that of the reaction solution and being immiscible with the reaction solution;
a first heating unit that heats a first region of the channel when the reaction container is attached to the attachment unit;
a second heating unit that heats a second region of the channel different from the first region when the reaction container is attached to the attachment unit;
a drive mechanism that switches arrangement of the attachment unit, the first heating unit, and the second heating unit between a first arrangement in which a lowermost position of the channel in a direction in which gravity acts is located within the first region and a second arrangement in which the lowermost position of the channel in the direction in which the gravity acts is located within the second region when the reaction container is attached to the attachment unit;
a measurement unit that measures the intensity of the light having the predetermined wavelength; and
a control unit that controls the drive mechanism, the first heating unit, the second heating unit, and the measurement unit,
wherein the control unit is capable of
first processing of controlling the first heating unit at a first temperature,
second processing of controlling the second heating unit at a second temperature higher than the first temperature,
third processing of controlling the drive mechanism to switch the arrangement of the attachment unit, the first heating unit, and the second heating unit from the second arrangement to the first arrangement if a first period has elapsed with the arrangement of the attachment unit, the first heating unit, and the second heating unit being the second arrangement, and
fourth processing of controlling the measurement unit to measure the intensity of the light having the predetermined wavelength after the third processing.

2. The thermal cycler according to claim 1, wherein the control unit is further capable of
fifth processing of allowing a second period to elapse with the arrangement of the attachment unit, the first heating unit, and the second heating unit being the second arrangement after the second processing, and
third processing after the fifth processing.

3. The thermal cycler according to claim 2, wherein the control unit is further capable of
sixth processing of controlling the first heating unit at a third temperature lower than the first temperature and allowing a third period to elapse with the arrangement of the attachment unit, the first heating unit, and the second heating unit being the first arrangement,
seventh processing of controlling the drive mechanism to switch the arrangement of the attachment unit, the first heating unit, and the second heating unit from the first arrangement to the second arrangement after the sixth processing, and
fifth processing after the seventh processing.

4. The thermal cycler according to claim 1, wherein the control unit performs eighth processing of controlling the drive mechanism to switch the arrangement of the attachment unit, the first heating unit, and the second heating unit from the first arrangement to the second arrangement if a fourth period has elapsed with the arrangement of the attachment unit, the first heating unit, and the second heating unit being the first arrangement, the third processing, and the fourth processing repeatedly at a predetermined number of times after the fourth processing.

5. The thermal cycler according to claim 1, wherein the measurement unit measures intensity of light from a region containing the first region.

6. A control method of a thermal cycler, including
an attachment unit for attachment of a removable reaction container, the reaction container including a channel filled with a reaction solution containing a fluorescent probe that changes intensity of light having a predetermined wavelength by binding to a DNA sequence and a liquid having a specific gravity different from that of the reaction solution and being immiscible with the reaction solution,
a first heating unit that heats a first region of the channel when the reaction container is attached to the attachment unit,
a second heating unit that heats a second region of the channel different from the first region when the reaction container is attached to the attachment unit,
a drive mechanism that switches arrangement of the attachment unit, the first heating unit, and the second heating unit between a first arrangement in which a lowermost position of the channel in a direction in which gravity acts is located within the first region and a second arrangement in which the lowermost position of the channel in the direction in which the gravity acts is located within the second region when the reaction container is attached to the attachment unit, and
a measurement unit that measures the intensity of the light having the predetermined wavelength,
the control method comprising:
performing first processing of controlling the first heating unit at a first temperature;
performing second processing of controlling the second heating unit at a second temperature higher than the first temperature;
performing third processing of controlling the drive mechanism to switch the arrangement of the attachment unit, the first heating unit, and the second heating unit from the second arrangement to the first arrangement if a first period has elapsed with the arrangement of the attachment unit, the first heating unit, and the second heating unit being the second arrangement; and
performing fourth processing of controlling the measurement unit to measure the intensity of the light having the predetermined wavelength after the third processing.