WO2020129116A1

WO2020129116A1 - Reaction treatment device, reaction treatment vessel, and reaction treatment method

Info

Publication number: WO2020129116A1
Application number: PCT/JP2018/046291
Authority: WO
Inventors: 福澤　隆; 磨川口
Original assignee: 日本板硝子株式会社
Priority date: 2018-12-17
Filing date: 2018-12-17
Publication date: 2020-06-25
Also published as: AR117328A1

Abstract

This reaction treatment device comprises: a reaction treatment vessel 10 comprising a flow path 12 through which a sample moves, a first filter 28 disposed on one end of the flow path 12, a second filter 30 disposed on another end of the flow path 12, a high-temperature region 36 provided on the one end side of the flow path 12, and a low-temperature region 38 provided on the another end side of the flow path 12; a temperature control system for keeping the high-temperature region 36 at a high temperature and keeping the medium-temperature region 38 at a medium temperature; and a fluid delivery system for moving the sample within the flow path 12. The first filter 28 and the second filter 30 are air permeable and water repellent. When the sample is moved from the high-temperature region 36 to the medium-temperature region 38, the sample is blocked by the second filter 30, whereby the sample is stopped in the medium-temperature region 38. When the sample is moved from the medium-temperature region 38 to the high-temperature region 36, the sample is blocked by the first filter 28, whereby the sample is stopped in the high-temperature region 36.

Description

Reaction processing apparatus, reaction processing container, and reaction processing method

The present invention relates to a reaction processing device, a reaction processing container, and a reaction processing method used in a polymerase chain reaction (PCR).

Genetic tests are widely used for tests in various medical fields, identification of crops and pathogenic microorganisms, food safety evaluation, and inspection for pathogenic viruses and various infectious diseases. In order to detect a minute amount of DNA with high sensitivity, a method of amplifying a part of DNA and analyzing the obtained product is known. Among them, the method using PCR is a technique of interest that selectively amplifies a portion containing a very small amount of DNA collected from a living body or the like.

In PCR, a predetermined thermal cycle is applied to a sample in which a biological sample containing DNA and a PCR reagent composed of a primer, an enzyme, etc. are subjected to repeated denaturation, annealing and extension reactions to cause a specific portion of DNA to be bound. It selectively amplifies.

In PCR, a target sample is generally put in a predetermined amount in a reaction treatment container such as a PCR tube or a microplate (microwell) having a plurality of holes formed therein, but recently, it was formed on a substrate. It has been put into practical use to carry out the reaction using a reaction processing container (also called a chip) having a fine channel (for example, Patent Document 1).

JP, 2009-232700, A

In PCR using a reciprocating flow channel type reaction container, in order to give a thermal cycle to a sample, a plurality of temperature regions maintained at different temperatures are set in the flow channel, and a plurality of temperature regions are set in the flow channel. Move back and forth. In order to properly subject the sample to thermal cycling, it is necessary for the sample to stop exactly in each temperature range. If the stop position varies, the reaction may not occur, the progress of the reaction may vary depending on the sample location, or the reaction such as DNA amplification may become inaccurate, which may lead to misjudgment by workers or workers. is there.

The present invention has been made in view of such circumstances, and an object thereof is to perform a reaction in which a sample can be subjected to a thermal cycle by reciprocally moving the sample within channels in which different temperature regions are set. It is an object of the present invention to provide a technique capable of accurately stopping a sample at a predetermined position in a temperature region in a processing device or a reaction processing container.

In order to solve the above-mentioned problems, a reaction treatment apparatus according to an aspect of the present invention is a channel in which a sample moves, a first filter disposed at one end of the channel, and a first filter disposed at the other end of the channel. 2 filter, a first temperature region provided on one end side of the flow channel, and a second temperature region provided on the other end side of the flow channel, and a first temperature region of the reaction treatment container A temperature control system that maintains the second temperature region of the reaction treatment container at a second temperature lower than the first temperature while maintaining the temperature at one temperature, and a liquid feeding system that moves the sample in the channel. The first filter and the second filter have air permeability and water repellency. When moving the sample from the first temperature region to the second temperature region by the liquid feeding system, the sample is stopped by the second filter, so that the sample stops at the second temperature region, and the liquid feeding system causes the second temperature region. When the sample is moved from the first temperature range to the first temperature range, the sample is stopped by the first filter, so that the sample stops in the first temperature range.

The first filter and the second filter may contain a fluororesin. The first filter and the second filter may contain at least one fluororesin selected from polytetrafluoroethylene, perfluoroalkoxyalkane, perfluoroethylenepropene copolymer, and ethylenetetrafluoroethylene copolymer.

The first filter and the second filter may be made of polytetrafluoroethylene.

The first temperature may be set to a temperature for denaturing the sample, and the second temperature may be set to a temperature for annealing and extending the sample. The reaction processing device may further include a fluorescence detector arranged to detect fluorescence from the sample located in a part of the second temperature region.

The reaction processing container may be provided with an annular packing to close the gap between the first filter and the flow passage and the gap between the second filter and the flow passage.

Another aspect of the present invention is to provide a flow path through which a sample moves, a first filter arranged at one end of the flow path, a second filter arranged at the other end of the flow path, and one end side of the flow path. And a second temperature region provided on the other end side of the flow path. The first filter and the second filter have air permeability and water repellency. When the sample is moved from the first temperature region to the second temperature region, the sample is stopped by the second filter, so that the sample stops in the second temperature region and changes from the second temperature region to the first temperature region. When the sample is moved, the sample is stopped by the first filter, so that the sample stops in the first temperature region.

Yet another aspect of the present invention is a flow path through which a sample moves, a first filter arranged at one end of the flow path, a second filter arranged at the other end of the flow path, and at one end side of the flow path. A reaction treatment container including a first temperature region provided and a second temperature region provided on the other end side of the flow path, and the reaction treatment container while maintaining the first temperature region of the reaction treatment container at the first temperature. Is a reaction processing method in a reaction processing apparatus, which includes a temperature control system for maintaining the second temperature region of the second temperature at a second temperature lower than the first temperature, and a liquid feeding system for moving the sample in the channel. In this method, the first filter and the second filter have air permeability and water repellency, and when the sample is moved from the first temperature region to the second temperature region by the liquid delivery system, The sample is stopped in the second temperature region by being blocked, and when the sample is moved by the liquid feeding system from the second temperature region to the first temperature region, the sample is blocked by the first filter, so that the sample Stop in the first temperature range.

The reaction processing method may be a real-time PCR method. The reaction treatment method may be an intercalator method.

The reaction treatment method may further include a melting analysis step. In the melting analysis step, the melting analysis curve may be obtained by changing the temperature within the second temperature region.

According to the present invention, in a reaction processing apparatus or reaction processing container capable of giving a thermal cycle to a sample by moving the sample in a reciprocating manner within channels in which different temperature regions are set, Can be accurately stopped at a predetermined position.

It is a top view of the substrate with which a reaction processing container is provided. It is a conceptual diagram for demonstrating the structure of a reaction processing container. It is a figure for demonstrating the cross-section of a reaction processing container. It is a schematic diagram for explaining the reaction processing apparatus concerning the embodiment of the present invention. It is a figure which shows the state in which the sample was sent into the intermediate temperature region of the flow path. It is a figure showing the state where the sample was sent into the high temperature field of a channel. It is a figure which shows the amplification result of PCR in a present Example. It is a figure which shows the melting analysis result in a present Example. It is a figure which shows the melting analysis result in a present Example. It is a conceptual diagram of the reaction processing container corresponding to the temperature range of three levels. It is a conceptual diagram of the reaction processing container used for the simultaneous reaction with respect to several samples.

The reaction processing device according to the embodiment of the present invention will be described below. The same or equivalent constituent elements, members, and processes shown in each drawing are denoted by the same reference numerals, and duplicated description will be omitted as appropriate. Further, the embodiments are merely examples and do not limit the invention, and all the features and combinations thereof described in the embodiments are not necessarily essential to the invention.

First, a reaction processing container usable in the reaction processing apparatus according to the embodiment of the present invention will be described. The reaction processing container includes a substrate, a sealing film attached to the substrate, and a filter. FIG. 1 is a plan view of a substrate included in the reaction processing container. FIG. 2 is a conceptual diagram for explaining the configuration of the reaction processing container. FIG. 3 is a diagram for explaining the cross-sectional structure of the reaction processing container. It should be noted that FIG. 3 is a diagram for explaining the positional relationship between the flow path formed on the substrate, the film, and the filter, and is different from the cross-sectional view of the reaction processing container of the embodiment.

The reaction processing container 10 includes a resinous substrate 14 having a groove-shaped channel 12 formed on an upper surface 14a, a channel sealing film 16 attached on the upper surface 14a of the substrate 14, a first sealing film 18, and The second sealing film 19, the third sealing film 20, the fourth sealing film 21, and the fifth sealing film 22 attached on the lower surface 14b of the substrate 14, and the first filter arranged in the substrate 14. 28 and the second filter 30. Further, an annular packing such as an O (O) ring 23 is provided between the first filter 28 and the third sealing film 20 and between the second filter 30 and the fourth sealing film 21. The O-ring 23 may be arranged so as not to move easily by pressing the filter against the substrate, and it is also possible to close the gap between the filter and the flow path. The annular packing has a shape corresponding to the shape of the filter or flow path to be used or the portion where the filter is installed, and may be a polygonal shape such as a substantially circular shape or a square shape.

The substrate 14 is preferably formed of a material that is stable against temperature changes and is not easily attacked by the sample solution used. Furthermore, the substrate 14 is preferably formed of a material having good moldability, good transparency and barrier properties, and low autofluorescence. As such a material, inorganic materials such as glass and silicon (Si), as well as resins such as acrylic, polypropylene, and silicone, among which cycloolefin polymer resin (COP) is preferable.

A groove-shaped channel 12 is formed on the upper surface 14a of the substrate 14. In the reaction processing container 10, most of the flow path 12 is formed in a groove shape exposed on the upper surface 14 a of the substrate 14. This is because it can be easily molded by injection molding using a mold or the like. In order to seal this groove and utilize it as a flow path, a flow path sealing film 16 is attached on the upper surface 14a of the substrate 14. The cross-sectional shape of the groove is not particularly limited and may be rectangular or U-shaped (round). Further, in order to improve the releasability at the time of molding, the shape may be tapered from the upper surface 14a in the depth direction, and may be trapezoidal, for example. An example of the dimensions of the flow channel 12 is 0.7 mm in width and 0.7 mm in depth.

One main surface of the flow channel sealing film 16 may have adhesiveness, or a functional layer exhibiting adhesiveness or adhesiveness by pressing, irradiation with energy such as ultraviolet rays, heating, or the like is provided on one main surface. It may be formed, and has a function of easily adhering to and integrating with the upper surface 14a of the substrate 14. The flow channel sealing film 16 is preferably formed of a material having low autofluorescence including an adhesive. In this respect, a transparent film made of a resin such as cycloolefin polymer, polyester, polypropylene, polyethylene or acryl is suitable, but not limited thereto. Further, the flow path sealing film 16 may be formed of plate-shaped glass or resin. In this case, since the rigid property can be expected, it is useful for preventing the reaction processing container 10 from being warped or deformed.

A first filter 28 is provided at one end 12 a of the flow path 12. A second filter 30 is provided at the other end 12b of the flow path 12. The pair of the first filter 28 and the second filter 30 provided at both ends of the flow channel 12 do not interfere with the amplification and detection of the target DNA by PCR or prevent the quality of the target DNA from deteriorating. Prevent contamination. The dimensions of the first filter 28 and the second filter 30 are formed so that they can fit in the filter installation space formed on the substrate 14 without any gap.

The substrate 14 is formed with a first air communication port 24 that communicates with the one end 12 a of the flow path 12 via the first filter 28. Similarly, the substrate 14 is formed with a second air communication port 26 that communicates with the other end 12b of the flow path 12 via the second filter 30. The pair of first air communication port 24 and second air communication port 26 are formed so as to be exposed on the upper surface 14 a of the substrate 14.

In the present embodiment, as the first filter 28 and the second filter 30, those having good low impurity characteristics and having air permeability and water repellency or oil repellency are used. As the first filter 28 and the second filter 30, a fluorine-containing resin is preferable, but not limited to these, PTFE (polytetrafluoroethylene), PFA (perfluoroalkoxyalkane), FEP (perfluoroethylenepropene copolymer), Examples thereof include ETFE (ethylene tetrafluoroethylene copolymer). Further, as the PTFE (polytetrafluoroethylene) filter, PF060 and PF100 (both manufactured by Advantech Group) can be used, although not limited thereto. PF060 has a thickness of 0.50 mm, a porosity of 75%, a retained particle size of 6 μm, a pressure loss of 0.069 kPa, and a water repellency of more than 3.9 kPa. The PF100 has a thickness of 1.0 mm, a porosity of 77%, a retained particle size of 10 μm, a pressure loss of 0.059 kPa, and a water repellency of more than 3.9 kPa. Further, as the first filter 28 and the second filter 30, those having a surface coated with a fluorine-containing resin and a water-repellent treatment can be used.

The flow path 12 is provided with a reaction region between a pair of the first filter 28 and the second filter 30 in which a plurality of levels of temperature can be controlled by a reaction processing device described later. A thermal cycle can be applied to the sample by moving the sample so as to continuously reciprocate in the reaction region in which the temperature of a plurality of levels is maintained.

In the present embodiment, the reaction region includes a high temperature region 36 provided on the one end 12a side of the flow channel 12 and a medium temperature region 38 provided on the other end 12b side of the flow channel 12. When the reaction processing container 10 is mounted on the reaction processing apparatus described later, the high temperature region 36 is maintained at a relatively high temperature (for example, about 95° C.), and the medium temperature region 38 is lower than the high temperature region 36 (for example, about 62° C.). ) Is maintained. The high temperature region 36 is located on the right side of the flow path 12 in the drawing, one end of which communicates with the first air communication port 24 through the first filter 28 and the connection flow path 29, and the other end of which connects with the connection flow path 40. It communicates with the intermediate temperature region 38 via the. The middle temperature region 38 is located on the left side of the flow path 12 in the drawing, one end thereof communicates with the high temperature region 36 via the connection flow path 40, and the other end thereof via the second filter 30 and the connection flow path 31. 2 It communicates with the air communication port 26.

Each of the high temperature region 36 and the medium temperature region 38 includes a meandering flow path in which a curved portion and a straight portion are combined and which are continuously folded back. With such a meandering flow path, it is possible to effectively use a limited effective area such as a heater that constitutes the temperature control system described later, and it is easy to reduce the temperature variation in the reaction region. In addition, there is an advantage that the substantial size of the reaction processing container can be reduced, which contributes to downsizing of the reaction processing apparatus. On the other hand, the connection flow passage 40 between the high temperature region 36 and the intermediate temperature region 38 may be a linear flow passage.

A branch point 40a is provided in the middle of the connection flow channel 40 between the high temperature region 36 and the medium temperature region 38, and a branch flow channel 42 branches from the branch point 40a. A sample introduction port 44 is formed at the tip of the branch channel 42 so as to be exposed on the lower surface 14 b of the substrate 14.

The first sealing film 18 is attached to the upper surface 14a of the substrate 14 so as to seal the first air communication port 24. The second sealing film 19 is attached to the upper surface 14a of the substrate 14 so as to seal the second air communication port 26. The third sealing film 20 is attached to the lower surface 14b of the substrate 14 so as to seal the connection channel 29 and the first filter 28. The fourth sealing film 21 is attached to the lower surface 14b of the substrate 14 so as to seal the connection channel 31 and the second filter 30. The fifth sealing film 22 is attached to the lower surface 14b of the substrate 14 so as to seal the sample introduction port 44. As these sealing films, a transparent film having a resin such as cycloolefin polymer, polyester, polypropylene, polyethylene or acrylic as a base material can be used. In the state where all the sealing films including the channel sealing film 16 are attached, all the channels are closed spaces.

When connecting the liquid supply system described later to the reaction treatment container 10, the first sealing film 18 and the second sealing film 19 which seal the first air communication port 24 and the second air communication port 26 are used. Peeling off and connecting the tube provided in the liquid feeding system to the first air communication port 24 and the second air communication port 26. Alternatively, the first sealing film 18 and the second sealing film 19 may be perforated with a hollow needle (a sharp-pointed injection needle) provided in the liquid delivery system. In this case, the first sealing film 18 and the second sealing film 19 are preferably films made of a material and a thickness that facilitates perforation with a needle.

The sample is introduced into the flow channel 12 through the sample introduction port 44 by once peeling off the fifth sealing film 22 from the substrate 14, and after introducing a predetermined amount of the sample, the fifth sealing film 22 is again removed from the substrate 14. It is attached back to the lower surface 14b. Therefore, as the fifth sealing film 22, it is desirable to use a film having an adhesive property such that it can withstand attachment/detachment for several cycles. Further, the fifth sealing film 22 may have a mode in which a new film is attached after the sample is introduced, and in this case, the importance of the characteristics regarding repeated attachment/detachment can be eased.

The method of introducing the sample into the sample introduction port 44 is not particularly limited, but an appropriate amount of sample may be directly introduced from the sample introduction port 44 with a pipette, a dropper, a syringe, or the like. Alternatively, the introduction method may be performed while preventing contamination through a cone-shaped needle tip having a built-in filter made of porous PTFE or polyethylene. Many types of such needle tips are generally sold and easily available, and can be attached to the tip of a pipette, a dropper, a syringe or the like for use. Further, after the sample is discharged and introduced by a pipette, a dropper, a syringe or the like, the sample may be moved to a predetermined position in the channel 12 by further pressing and pushing.

Examples of the sample include, for example, a mixture containing one or two or more kinds of DNA, to which a thermostable enzyme and four kinds of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP) were added as PCR reagents. To be Further, a primer that specifically reacts with the DNA to be subjected to the reaction treatment, and in some cases, a fluorescent probe such as TaqMan (TaqMan is a registered trademark of Roche Diagnostic Geselle Shaft Mitt Beschlenktel Haftung) for TaqMan or SYBR Green (SYBR is a molecular probe). S. Inc.'s registered trademark). A commercially available real-time PCR reagent kit or the like can also be used.

FIG. 4 is a schematic diagram for explaining the reaction processing device 100 according to the embodiment of the present invention.

The reaction processing device 100 according to the present embodiment includes a reaction processing container mounting portion (not shown) on which the reaction processing container 10 is mounted, a temperature control system 50, and a CPU 52. The temperature control system 50 has a high temperature region in the flow path 12 of the reaction treatment container 10 of about 95° C. (high temperature region) and a middle temperature region of about 62 with respect to the reaction treatment container 10 placed on the reaction treatment container placement portion. It is constructed so that it can be accurately maintained and controlled at ℃.

The temperature control system 50 maintains the temperature of each temperature region of the reaction region, and specifically, the high temperature heater 54 for heating the high temperature region 36 of the flow passage 12, and the medium temperature of the flow passage 12. A low temperature heater 56 for heating the region 38, a temperature sensor (not shown) such as a thermocouple for measuring the actual temperature of each temperature region, and a high temperature heater driver for controlling the temperature of the high temperature heater 54. 58 and a low temperature heater driver 60 that controls the temperature of the low temperature heater 56. The actual temperature information measured by the temperature sensor is sent to the CPU 52. The CPU 52 controls each heater driver so that the temperature of each heater becomes a predetermined temperature based on the actual temperature information of each temperature region. Each heater may be, for example, a resistance heating element or a Peltier element. The temperature control system 50 may further include other components for improving temperature controllability in each temperature range.

The reaction processing apparatus 100 according to the present embodiment further includes a liquid delivery system 62 for moving the sample S introduced into the channel 12 of the reaction processing container 10 in the channel 12. The liquid delivery system 62 includes a first pump 64, a second pump 66, a first pump driver 68 for driving the first pump 64, a second pump driver 70 for driving the second pump 66, A first tube 72 and a second tube 74 are provided.

One end of the first tube 72 is connected to the first air communication port 24 of the reaction processing container 10. A packing 76 or a seal for ensuring airtightness is preferably arranged at a connection portion between the first air communication port 24 and one end of the first tube 72. The other end of the first tube 72 is connected to the output of the first pump 64. Similarly, one end of the second tube 74 is connected to the second air communication port 26 of the reaction processing container 10. A packing 77 or a seal for ensuring airtightness is preferably arranged at a connection portion between the second air communication port 26 and one end of the second tube 74. The other end of the second tube 74 is connected to the output of the second pump 66.

The first pump 64 and the second pump 66 may be, for example, a blower pump including a diaphragm pump, a micro blower pump, or a blower. Such blower pumps, micro blower pumps, and blowers can increase the pressure on the secondary side from the primary side during operation, but the characteristics are such that the pressure on the primary side becomes equal to the pressure on the secondary side at the moment of stopping or at the time of stopping. Is preferably provided. This is because, for example, when the sample obtains a propulsive force due to its blowing air or pressure, the propulsive force can be eliminated at the moment the pump stops, and the sample stop control becomes easy. ..

The CPU 52 controls air blow and pressurization from the first pump 64 and the second pump 66 via the first pump driver 68 and the second pump driver 70. The air blow and pressurization from the first pump 64 and the second pump 66 act on the sample S in the flow path 12 through the first air communication port 24 and the second air communication port 26, and become a propulsive force to the sample S. To move. More specifically, by alternately operating the first pump 64 and the second pump 66, the pressure applied to one of the end faces of the sample S becomes larger than the pressure applied to the other end thereof, so that the propulsion related to the movement of the sample S is promoted. Power is gained. By alternately operating the first pump 64 and the second pump 66, the sample S can be reciprocally moved in the flow path and passed through each temperature region of the flow path 12 of the reaction processing container 10, As a result, it becomes possible to apply a thermal cycle to the sample S. More specifically, by repeatedly applying the steps of denaturation in the high temperature region 36 and annealing/extension in the intermediate temperature region 38, the target DNA in the sample S is selectively amplified. In other words, the high temperature region 36 can be regarded as the denaturing temperature region, and the medium temperature region 38 can be regarded as the annealing/extension temperature region. Further, the residence time in each temperature region can be appropriately set by changing the time when the sample S stops at a predetermined position in each temperature region.

The reaction processing device 100 according to the present embodiment further includes a fluorescence detector 78. Generally, in real-time PCR, a reagent that emits fluorescence is added to a sample. Since the intensity of the fluorescence signal emitted from the sample increases as the amplification of DNA progresses, the intensity value of the fluorescence signal can be used as an index as a judgment material for the progress of PCR and the end of the reaction.

As the fluorescence detector 78, an optical fiber type fluorescence detection manufactured by Nippon Sheet Glass Co., Ltd., which is a very compact optical system, can perform rapid measurement, and can detect fluorescence regardless of whether it is a bright place or a dark place. The vessel FLE-510 can be used. This optical fiber type fluorescence detector can be tuned so that the wavelength characteristic of the excitation light/fluorescence is suitable for the fluorescence characteristic emitted by the sample, and provides an optimum optical detection system for the sample having various characteristics. It is also possible to detect fluorescence from a sample existing in a small or narrow area such as a channel with a small noise because of the small diameter of the light beam provided by the optical fiber type fluorescence detector. ..

The optical fiber type fluorescence detector 78 includes an optical head 80, a fluorescence detector driver 82, and an optical fiber 84 connecting the optical head 80 and the fluorescence detector driver 82. The fluorescence detector driver 82 includes an excitation light source (LED, laser, or other light source adjusted to emit a specific wavelength), an optical fiber type multiplexer/demultiplexer, and a photoelectric conversion element (PD, APD, Photomal, etc.). A photodetector) (neither of which is shown) and the like are included, and a driver and the like for controlling these are included. The optical head 80 is composed of an optical system such as a lens, and has a function of directionally irradiating the sample S with excitation light and a function of condensing fluorescence emitted from the sample. The collected fluorescence is separated from the excitation light by the optical fiber type multiplexer/demultiplexer in the fluorescence detector driver 82 through the optical fiber 84, and is converted into an electric signal by the photoelectric conversion element. The fluorescence detector is not limited to the optical fiber type fluorescence detector as long as it has a function of detecting the fluorescence from the sample S.

In the reaction processing apparatus 100 according to the present embodiment, as long as the progress of amplification of DNA contained in the sample in the channel can be known, the position of the fluorescence detection region is in any of the regions along the channel. May be. For example, the optical head 80 may be arranged so that fluorescence can be detected from the sample S located in a partial area 86 (referred to as “fluorescence detection area 86”) in the intermediate temperature area 38. The reaction of the sample S progresses as the sample S is repeatedly moved back and forth in the channel, and a predetermined DNA contained in the sample S is amplified. Therefore, the progress of the amplification of the DNA is monitored by monitoring the fluctuation of the detected fluorescence amount. You can know in real time.

Next, the operation of applying a thermal cycle to the sample S by the reaction processing device 100 will be described.

First, according to the following procedures (1) to (6), the reaction processing container 10 is filled with the sample S, and the reaction processing container 10 is set in the reaction processing apparatus 100. Further, before setting the reaction processing container 10 in the reaction processing apparatus 100, the first pump 64 and the second pump 66 are operated to operate the air such as the

packings

76 and 77 in addition to the first tube 72 and the second tube 74. The path passing through may be blown with air. By doing so, aerosols and the like that may remain in the path can be eliminated, which helps prevent carryover.
(1) Either one of the first sealing film 18 that seals the first air communication port 24 and the second sealing film 19 that seals the second air communication port 26 is peeled off.
(2) The fifth sealing film 22 that seals the sample introduction port 44 is peeled off.
(3) The sample S is introduced from the sample introduction port 44 with a pipette or the like, and the sample S is pushed to the tip of the branch point 40a.
(4) The fifth sealing film 22 is attached back. (5) The other of the first sealing film 18 and the second sealing film 19 is peeled off.
(6) The reaction processing container 10 is arranged in the reaction processing container mounting portion of the reaction processing apparatus 100, and the first tube 72 and the second tube of the liquid delivery system 62 are connected to the first air communication port 24 and the second air communication port 26. 74 is connected.

Next, the reaction processing apparatus 100 is operated according to the following procedures (7) to (11).
(7) FIG. 5 shows a state in which the sample S is sent to the medium temperature region 38 of the flow channel 12. For example, the pressure of the flow path 12 communicating with the first air communication port 24 is set higher than the pressure of the flow path 12 communicating with the second air communication port 26. Alternatively, air is sent in through the first air communication port 24. As a result, the sample S is sent to the intermediate temperature region 38 of the flow path 12. The sample S sent to the intermediate temperature region 38 is blocked by the second filter 30 as shown in FIG. This is because the second filter 30 has air permeability and water repellency.
(8) With the sample S stopped in the medium temperature region 38 of the flow channel 12, wait until the medium temperature region 38 and the high temperature region 36 of the flow channel 12 reach a predetermined temperature. For example, the medium temperature region 38 has a temperature (for example, about 62° C.) at which the sample S is annealed and extended, and the high temperature region 36 has a temperature (for example, about 95° C.) at which the sample S is denatured.
(9) FIG. 6 shows a state in which the sample S is sent to the high temperature region 36 of the flow channel 12. After the intermediate temperature region 38 and the high temperature region 36 reach a predetermined temperature, the reaction processing apparatus 100 is operated to send the sample S into the high temperature region 36 of the flow path 12. For example, the pressure of the flow path 12 communicating with the second air communication port 26 is set higher than the pressure of the flow path 12 communicating with the first air communication port 24. Alternatively, air is sent in through the second air communication port 26. As a result, the sample S is sent to the high temperature region 36 of the flow channel 12. The sample S sent to the high temperature region 36 is blocked by the first filter 28 as shown in FIG. This is because the first filter 28 has air permeability and water repellency.
(10) After the predetermined reaction time in the high temperature region 36 has elapsed, the sample S is sent into the intermediate temperature region 38 again.
(11) By repeating the reciprocating movement between the high temperature region 36 and the intermediate temperature region 38 a predetermined number of times, the thermal cycle can be applied to the sample S and PCR can be performed. When the sample S is in the intermediate temperature region 38, the fluorescence from the sample S located in the fluorescence detection region 86 is detected, and the fluorescence intensity is monitored to measure the progress of PCR along with the thermal cycle. can do.

As described above, in the reaction processing apparatus 100 according to this embodiment, a filter having air permeability and water repellency is used as the filter of the reaction processing container 10, and the flow path 12 of the reaction processing container 10 is used by the liquid delivery system 62. When the sample S was moved inside, the sample S was stopped in each temperature region by blocking the sample S with a filter. Since the stop position of the sample S is determined by the filter, the sample S can be accurately stopped at a predetermined position in each temperature region.

According to the present embodiment, for example, the stop position of the sample S is detected by the filter rather than the configuration and processing such that the position of the sample S is detected by the fluorescence detector and the liquid delivery system 62 is controlled based on the detection result. Since it is mechanically determined, the apparatus configuration can be simplified and the cost of the reaction processing apparatus 100 can be reduced. The reason why the first filter 28 and the second filter 30 are required to have water repellency or oil repellency is that when the samples come in contact with the respective filters and are dammed, the liquid sample infiltrates into the filter medium or a part of the sample is removed. This is to prevent the filter from passing through.

When variations occur in the stop position of the sample S, it is necessary to widen each temperature region to absorb the variations, and as a result, the reaction processing apparatus 100 may become large. However, in the reaction processing apparatus 100 according to the present embodiment, since the sample S always stops at the filter position, it is not necessary to make each temperature region wider than necessary as compared with the case where the stop position has variations. Further, the size of the heater for heating each temperature region can be reduced. Therefore, according to the present embodiment, the reaction processing device 100 can be downsized. Further, the downsizing of the heater can reduce power consumption.

Generally, the real-time PCR method is roughly divided into a probe method and an intercalator method. The probe method requires a primer specific to the target DNA sequence and a (fluorescent) probe for amplification, and the fluorescence signal increases with amplification as the probe decomposes. On the other hand, in the intercalator method, a fluorescent dye (eg, SYBR Green) that is incorporated into a DNA double strand to generate fluorescence is mixed without using a probe, and the DNA double strand increases with amplification, and a fluorescent signal is proportionally generated. Will increase. However, in the intercalator method, the number of unique ones in the DNA sequence of interest, such as in the probe method, decreases, so that nonspecific reaction may increase, and it is necessary to confirm whether or not this nonspecific reaction occurs. In the intercalator method, "melting analysis" is usually performed to confirm the presence or absence of such nonspecific reaction.

In melting analysis, the relationship between temperature and fluorescence intensity is acquired for the sample after PCR and graphed. Since the DNA double strand is decomposed at a predetermined temperature (depending on the length of DNA, etc.) as the temperature is raised, the fluorescence intensity rapidly changes at this temperature. In other words, if there is only one peak in the differential curve (called "melting analysis curve") of this graph, it can be seen that there was no nonspecific reaction.

The reaction processing apparatus 100 according to this embodiment can apply both the probe method and the intercalator method. In the intercalator method, when DNA contained in a sample undergoes thermal denaturation in a high temperature region, the sample does not emit fluorescence. Therefore, when the fluorescence detection region is provided only at the location corresponding to the connection flow path between the high temperature region and the medium temperature region, the fluorescence emitted from the sample cannot be properly detected. Therefore, when controlling the position and speed of the sample based only on the fluorescence signal from the fluorescence detector corresponding to the fluorescence detection region, there is a possibility that it may interfere with proper reciprocal movement and stop of the sample. ..
In the reaction processing device 100 according to the present embodiment, as described above, the sample is configured to stop at the position of the filter, and the fluorescence detection region is in any of the regions along the flow path, Unless the position and speed of the sample are controlled based only on the fluorescence signal from the fluorescence detector, there is little probability that it will hinder the proper reciprocal movement and stop of the sample.

Further, in the reaction processing apparatus 100 according to the present embodiment, when the fluorescence detection region 86 is provided in the intermediate temperature region 38, DNA is amplified by detecting fluorescence from the sample in the process of reciprocating movement of the sample. In addition to being able to know the progress of the sample in real time, the fluorescence intensity can be measured by changing the temperature of the flow path in this region with the sample stopped in the intermediate temperature region 38 after the predetermined number of reciprocating movements and the end of the thermal cycle. Can be measured. This can be utilized, for example, when performing melting analysis. Further, since it is not necessary to add a separate configuration for that purpose, the melting analysis after amplification necessary for PCR by the intercalator method can be easily performed. Therefore, when the intercalator method involving melting analysis is performed in addition to the probe method, it is desirable that the fluorescence detection region be provided in the intermediate temperature region 38. Since the melting analysis includes the step of measuring the intensity of the fluorescence signal as the temperature rises, it is preferable to perform it in the middle temperature region 38 where the temperature is relatively low from the beginning.

Further, in the reaction processing device 100 according to the present embodiment, a fluorescence detection region may be provided at a location corresponding to the connection flow path between the high temperature region 36 and the intermediate temperature region 38. When PCR is performed by the probe method, fluorescence is emitted from the sample even in the connection flow channel, so by detecting this fluorescence signal, the position and speed of the sample can be identified based on the fluorescence signal from the fluorescence detector. Can contribute to.

Further, in the reaction processing device 100 according to this embodiment, a fluorescence detection region may be provided in the high temperature region 36. When PCR is performed by the probe method, fluorescence is emitted from the sample even in the high temperature region. Therefore, by detecting this fluorescence signal, it can be understood that the sample exists in the high temperature region. This makes it possible to detect the state where the sample has entered each temperature region, which suggests that it contributes to more appropriate reciprocating movement of the sample, including highly accurate control of the stop position.

Next, examples of the present invention will be described. Using the above-described reaction processing apparatus 100 having the fluorescence detection region 86 in the

medium temperature region

38, 1 μL of 1 μL template 1×10 ⁵ Copies/μL was added to ¹⁵ μL of the sample shown in Table 1, and further, Takara Bio's Real-time PCR was carried out by using a 1000-fold diluted solution of SYBR Green.

FIG. 7 shows the result of PCR amplification in this example. In FIG. 7, the horizontal axis represents the number of cycles and the vertical axis represents the fluorescence intensity [arbitrary unit]. As shown in FIG. 7, the intensity of the fluorescence signal increases with the number of PCR cycles. The intensity of the fluorescence signal increased around the number of cycles exceeding 18 and remarkable amplification started, and a plateau state was reached around the number of cycles exceeding 28. Such an amplification curve of the fluorescence signal shows that the sample in the sample is amplified, and it can be seen that good PCR can be performed using the reaction processing device 100 according to the present embodiment.

8 and 9 are diagrams showing melting analysis results in this example. After the real-time PCR was performed, the sample was moved to the intermediate temperature region 38, and the fluorescence signal of the sample located in the fluorescence detection region 86 was continuously measured while increasing the temperature in the intermediate temperature region 38. FIG. 8 shows the relationship between the fluorescence signal intensity and the temperature in the intermediate temperature region 38. FIG. 9 is a so-called melting analysis curve, which is obtained by differentiating the curve shown in FIG. 8 once.

From FIG. 8, it can be seen that the fluorescence intensity decreases as the temperature rises, and decreases more sharply after exceeding 87° C., and the influence of heat denaturation increases. Further, it can be seen from FIG. 8 that the fluorescence intensity tends to decrease and stop around around 93° C.

From FIG. 9, it is clear that the melting analysis curve has only one peak at 88.4°C. It can be seen that the PCR amplification product in this example does not include a relatively short amplification product such as Primer Dimer.

A modification of the reaction processing container will be described below.

FIG. 10 is a conceptual diagram of the reaction processing container 110 corresponding to the three-level temperature regions. Although the reaction processing container 10 having two-level reaction regions of high temperature and medium temperature has been described above, the reaction processing container 110 shown in FIG. 10 has three-level reaction regions of high temperature, medium temperature and low temperature.

The reaction processing container 110 includes a first flow channel 112a, a second flow channel 112b, and a third flow channel 112c that extend in three directions from a branch portion 112d. The first filter 128 is arranged at one end of the first flow passage 112a, the second filter 130 is arranged at one end of the second flow passage 112b, and the third filter 131 is arranged at one end of the third flow passage 112c. ing. The first filter 128 is in communication with the first air communication port 124, the second filter 130 is in communication with the second air communication port 126, and the third filter 131 is in communication with the third air communication port 127. ing. The first flow passage 112a is provided with a high temperature region 136, the second flow passage 112b is provided with a medium temperature region 138, and the third flow passage 112c is provided with a low temperature region 139. A branch point 142a is provided in the middle of the third flow path 112c, and the branch flow path 142 branches from the branch point 142a. A sample introduction port 144 is provided at the tip of the branch channel 142.

Also in the reaction processing container 110 configured as described above, when the sample is moved to each temperature region by the liquid feeding system of the reaction processing device, the sample is stopped in each temperature region by blocking the sample with the filter. .. That is, when the sample moves to the high temperature region 136, the first filter 128 blocks the sample. When the sample moves to the intermediate temperature region 138, the second filter 130 blocks the sample. When the sample moves to the low temperature region 139, the sample is blocked by the third filter 131. Since the stop position of the sample is determined by the filter, the sample can be accurately stopped at a predetermined position in each temperature region.

Also, in the reaction processing container 110, a fluorescence detection region 186 is provided in the medium temperature region 138. Thereby, not only the probe method but also the intercalator method can be suitably applied.

In a reaction processing device that uses the reaction processing container 110, a valve is added between the pump and the air communication port. For example, when the liquid is fed from the medium temperature region 138 to the high temperature region 136, the valve on the low temperature region 139 side is closed to allow the liquid to be fed from the medium temperature region 138 to the high temperature region 136 while preventing the sample from flowing into the low temperature region 139.

FIG. 11 is a conceptual diagram of a reaction processing container 210 provided for simultaneous reaction with respect to a plurality of samples. FIG. 11 shows, as an example, a reaction processing container 210 capable of simultaneously performing PCR on two samples, but an arbitrary number of samples can be handled by changing the number of channels.

The reaction processing container 210 includes a first flow path 212a and a second flow path 212b arranged in parallel. The first filter 228 is arranged at one end of the first flow path 212a, and the second filter 230 is arranged at the other end. The third filter 229 is arranged at one end of the second flow path 212b, and the fourth filter 231 is arranged at the other end. A common first air communication port 224 communicates with the first filter 228 and the third filter 229. A common second air communication port 226 communicates with the second filter 230 and the fourth filter 231. A common high temperature region 236 is provided on one end side of the first flow path 212a and the second flow path 212b. A common intermediate temperature region 238 is provided on the other end sides of the first flow path 212a and the second flow path 212b. A first branch point 242a is provided in the middle of the first channel 212a, and the first branch channel 242 branches from the first branch point 242a. A first sample introduction port 244 is provided at the tip of the first branch flow channel 242. Similarly, a second branch point 243a is provided in the middle of the second flow path 212b, and the second branch flow path 243 branches from the second branch point 243a. A second sample introduction port 245 is provided at the tip of the second branch flow channel 243.

Also in the reaction processing container 210 configured as described above, when the sample is moved to each temperature region by the liquid feeding system of the reaction processing device, the sample is stopped by the filter by blocking the sample with each filter. .. That is, when the first sample in the first channel 212a and the second sample in the second channel 212b move to the high temperature region 236, they are blocked by the first filter 228 and the third filter 229, respectively. Further, when the first sample in the first flow path 212a and the second sample in the second flow path 212b move to the intermediate temperature region 238, they are blocked by the second filter 230 and the fourth filter 231 respectively. Since the stop position of the sample is determined by the filter, the sample can be accurately stopped at a predetermined position in each temperature region.

With the reaction processing container 210, PCR can be simultaneously performed on two samples. Since only one liquid sending system is required for the two flow paths, it is possible to reduce the size and cost of the reaction processing apparatus that uses the reaction processing container 210.

Also in the reaction processing container 210, the first fluorescence detection region 286 is provided in the middle temperature region 238 of the first flow path 212a, and the second fluorescence detection region 287 is provided in the middle temperature region 238 of the second flow path 212b. Thereby, not only the probe method but also the intercalator method can be suitably applied.

Above, the present invention has been described based on the embodiments. This embodiment is an example, and it is understood by those skilled in the art that various modifications can be made to the combinations of the respective constituent elements and the respective processing processes, and such modifications are also within the scope of the present invention. is there.

10, 110, 210 reaction processing container, 12 channels, 14 substrates, 16 channels sealing film, 18 first sealing film, 19 second sealing film, 20 third sealing film, 21 fourth sealing film , 22, 5th sealing film, 23 O-ring, 24, 124, 224 1st air communication port, 26, 126, 226 2nd air communication port, 28, 128, 228 1st filter, 29, 31, 40 connection flow Road, 30, 130, 230 second filter, 36, 136, 236 high temperature area, 38, 138, 238 medium temperature area, 42, 142 branch flow path, 44, 144 sample introduction port, 50 temperature control system, 52 CPU, 54 High temperature heater, 56 low temperature heater, 58 high temperature heater driver, 60 low temperature heater driver, 62 liquid transfer system, 64 first pump, 66 second pump, 68 first pump driver, 70 second pump driver, 72 second pump driver 1 tube, 74 second tube, 76, 77 packing, 78 fluorescence detector, 80 optical head, 82 fluorescence detector driver, 84 optical fiber, 86,186 fluorescence detection area, 100 reaction processing device, 127 third air communication port , 131 third filter, 139 low temperature region, 229 third filter, 231 fourth filter, 242 first branch flow channel, 243 second branch flow channel, 244 first sample introduction port, 245 second sample introduction port, 286 second number 1 fluorescence detection area, 287 2nd fluorescence detection area.

The present invention can be used for polymerase chain reaction (PCR).

Sequence number 1: Forward PCR primer Sequence number 2: Reverse PCR primer

Claims

A channel through which a sample moves, a first filter arranged at one end of the channel, a second filter arranged at the other end of the channel, and a first temperature provided at the one end side of the channel A reaction processing container including a region and a second temperature region provided on the other end side of the flow path,
A temperature control system for maintaining the first temperature region of the reaction treatment container at a first temperature and maintaining the second temperature region of the reaction treatment container at a second temperature lower than the first temperature;
A liquid transfer system for moving the sample in the channel,
Equipped with
The first filter and the second filter have air permeability and water repellency,
When the sample is moved from the first temperature region to the second temperature region by the liquid sending system, the sample is stopped by the second filter, so that the sample stops in the second temperature region,
When the sample is moved from the second temperature region to the first temperature region by the liquid delivery system, the sample is stopped by the first filter, so that the sample stops in the first temperature region. Characteristic reaction processing device.
The reaction processing apparatus according to claim 1, wherein the first filter and the second filter contain a fluororesin.
The first filter and the second filter contain at least one fluororesin selected from polytetrafluoroethylene, perfluoroalkoxyalkane, perfluoroethylenepropene copolymer, and ethylenetetrafluoroethylene copolymer. 2. The reaction processing apparatus according to item 2.
The reaction processing apparatus according to claim 3, wherein the first filter and the second filter are made of polytetrafluoroethylene.
The first temperature is set to a temperature for denaturing a sample, the second temperature is set to a temperature for annealing and extending the sample,
5. The reaction according to claim 1, further comprising a fluorescence detector arranged so as to detect fluorescence from the sample located in a part of the second temperature region. Processing equipment.
The reaction treatment container comprises an annular packing for closing a gap between the first filter and the flow passage and a gap between the second filter and the flow passage. 5. The reaction processing device according to any one of 5.
A channel through which a sample moves, a first filter arranged at one end of the channel, a second filter arranged at the other end of the channel, and a first temperature provided at the one end side of the channel A reaction processing container comprising a region and a second temperature region provided on the other end side of the flow path,
The first filter and the second filter have air permeability and water repellency,
When the sample is moved from the first temperature region to the second temperature region, the sample is stopped in the second temperature region by blocking the sample by the second filter,
When the sample is moved from the second temperature region to the first temperature region, the sample is blocked by the first filter, so that the sample stops in the first temperature region. Processing container.
The reaction processing container according to claim 7, wherein the first filter and the second filter contain a fluororesin.
The first filter and the second filter contain at least one fluororesin selected from polytetrafluoroethylene, perfluoroalkoxyalkane, perfluoroethylenepropene copolymer, and ethylenetetrafluoroethylene copolymer. 8. The reaction processing container according to item 8.
The reaction processing container according to claim 9, wherein the first filter and the second filter are made of polytetrafluoroethylene.
A channel through which a sample moves, a first filter arranged at one end of the channel, a second filter arranged at the other end of the channel, and a first temperature provided at the one end side of the channel A reaction processing container including a region and a second temperature region provided on the other end side of the flow path,
A temperature control system for maintaining the first temperature region of the reaction treatment container at a first temperature and maintaining the second temperature region of the reaction treatment container at a second temperature lower than the first temperature;
A liquid transfer system for moving the sample in the channel,
A reaction treatment method in a reaction treatment device comprising:
The first filter and the second filter have air permeability and water repellency,
When the sample is moved from the first temperature region to the second temperature region by the liquid sending system, the sample is stopped by the second filter, so that the sample stops in the second temperature region,
When the sample is moved from the second temperature region to the first temperature region by the liquid delivery system, the sample is stopped by the first filter, so that the sample stops in the first temperature region. Characterized reaction treatment method.
The reaction treatment method according to claim 11, wherein the first filter and the second filter contain a fluororesin.
The first filter and the second filter contain at least one fluororesin selected from polytetrafluoroethylene, perfluoroalkoxyalkane, perfluoroethylenepropene copolymer, and ethylenetetrafluoroethylene copolymer. 12. The reaction treatment method according to item 12.
The reaction treatment method according to claim 13, wherein the first filter and the second filter are made of polytetrafluoroethylene.
The reaction processing device further comprises a fluorescence detector,
The first temperature is set to a temperature for denaturing a sample, the second temperature is set to a temperature for annealing and extending the sample,
15. The reaction treatment method according to claim 11, wherein fluorescence from the sample located in a part of the second temperature region is detected using the fluorescence detector.
The reaction processing method according to any one of claims 11 to 15, wherein the reaction processing method is a real-time PCR method.
The reaction treatment method according to any one of claims 11 to 16, wherein the reaction treatment method is an intercalator method.
The reaction treatment method according to claim 17, further comprising a melting analysis step.
The reaction treatment method according to claim 18, wherein in the melting analysis step, a melting analysis curve is obtained by changing the temperature in the second temperature region.