Patent Description:
Genetic testing is widely used for examinations in a wide variety of medical fields, identification of farm products and pathogenic microorganisms, safety assessment for food products, and even for examinations for pathogenic viruses and a variety of infectious diseases. In order to detect with high sensitivity a minute amount of gene's DNA, methods of analyzing the resultant obtained by amplifying a portion of DNA are known. Above all, PCR is a remarkable technology where a certain portion of a very small amount of DNA collected from an organism or the like is selectively amplified.

In PCR, a predetermined thermal cycle is applied to a sample in which a biological sample containing DNA and a PCR reagent consisting of primers, enzymes, and the like are mixed so as to cause reactions such as denaturation, annealing, and elongation to be repeated so that a specific portion of DNA is selectively amplified.

It is a common practice to perform the processing of a reaction where a minute amount of sample is used such as PCR in a container called a vial or in a channel formed on a chip. For miniaturization and speeding up, technologies for performing PCR in a channel are sometimes advantageous, and many aspects thereof have been put to practical use.

In a thermal cycle of PCR, it is necessary to repeat a temperature cycle from a low temperature of at least about <NUM> to a high temperature of about <NUM> for a predetermined number of times for a sample in which DNA to be amplified and a PCR reagent are mixed. Since the sample is normally an aqueous solution, the vapor pressure becomes high in the <NUM> range, and the water content of the sample is likely to evaporate. If the water content of the sample evaporates, the concentration of the sample may become high, and parameters such as the optical characteristics of the sample may unexpectedly change, possibly causing problems such as not being able to properly manage the reaction processing step. Particularly in real-time PCR etc., since it is necessary to continually monitor the optical properties, etc., of the sample while performing PCR, there is a possibility that the progress of the reaction processing cannot be kept track of. Also, if bubbles are generated in the channel due to the evaporation of the sample, there is a possibility that the movement of the sample existing in the channel is prevented by the bubbles.

Particularly, when the place where PCR is performed is in an environment with low atmospheric pressure such as a high altitude place, this becomes a particularly apparent problem. In other words, since the atmospheric pressure decreases as the altitude increases, the boiling point drops remarkably in such an environment, causing the sample to boil easily. For example, the atmospheric pressure is roughly <NUM> hPa and the boiling point is <NUM> according to calculations at a place where the altitude is <NUM>, the atmospheric pressure is <NUM> hPa and the boiling point is <NUM> at a place where the altitude is <NUM>, and the atmospheric pressure is <NUM> hPa and the boiling point is <NUM> at a place where the altitude is <NUM>. Such high altitude places include Denver (altitude of about <NUM>), Mexico City (altitude of about <NUM>), etc. In such places, the sample boils easily in a high temperature range and thus vaporizes and/or foams, or the evaporation of the sample becomes remarkable; thus, it is difficult to practically perform PCR.

Also, the pressure inside a passenger airplane is about <NUM> hPa, which is equal to about <NUM> in altitude, and the boiling point is about <NUM>, meaning that it is practically difficult to perform PCR even inside an airplane in flight. This means that a circumstance occurs that becomes an obstacle for taking immediate preventive measures for recognizing the existence of viruses, pathogens, etc., in an airplane so as to prevent them before entering the country in order to prevent their worldwide spread. Also in flatlands, by raising the temperature of a sample mainly composed of an aqueous solution to around <NUM>, the sample is very likely to evaporate partially although the sample does not come to a boil, and a fluorescence signal may not be measured accurately in the case of performing PCR inside a channel.

In view of this, for the purpose of preventing a decrease in volume due to evaporation of a sample or the like, a configuration has been suggested in the related art where a liquid having a low vapor pressure (a high boiling point) such as oil is arranged at both ends of the sample so as to allow the liquid to function as a so-called "lid" (see, for example, Patent Document <NUM>). By putting a "lid" using a non-volatile liquid on both sides of the sample, the evaporation of the sample can be prevented.

Further, a structure has been proposed where air bubbles generated inside a channel are positively discharged from the channel by providing a gas hole, a hydrophobic filter, or the like in the channel (see, for example, Patent Document <NUM>).

However, as described in Patent Document <NUM>, it is very troublesome to prepare for a task of putting a lid using oil or the like in such a manner that a sample subjected to PCR is sandwiched, and there is also a problem in terms of preventing contamination of the sample. Furthermore, in an embodiment according to Patent Document <NUM>, it is considered that there is a case where boiling of a sample cannot be prevented under an environment where the boiling point of the sample becomes low.

Also, as described in Patent Document <NUM>, positively removing air bubbles from a channel is not a fundamental solution from the viewpoint of preventing a decrease in volume caused due to the boiling and/or evaporation of a sample, and, more than anything, the concentration of the sample may rise.

In this background, a purpose of the present invention is to provide a reaction processor, a reaction processing vessel, and a reaction processing method capable of performing PCR while preventing the boiling of a sample and the generation of air bubbles even in a place where the air pressure is low.

A reaction processor according to one embodiment of the present invention is defined in claim <NUM>. It includes: a placing portion for placing a plate-like reaction processing vessel that is provided with a channel into which a sample is introduced; a temperature control system that controls the temperature of a region in which the channel exists in order to heat the sample inside the channel; and a liquid feeding system that controls the pressure inside the channel of the reaction processing vessel so as to move the sample inside the channel. Further, the liquid feeding system maintains the pressure inside the channel during a reaction process of the sample to be higher than the air pressure in the surrounding environment of the reaction processor, preferably <NUM> atm or higher.

The liquid feeding system may include a pressurizing chamber that has an internal pressure maintained to be higher than the air pressure in the surrounding environment of the reaction processor, preferably <NUM> atm or higher; and a liquid feeding pump that is arranged inside the pressurizing chamber. The output of the liquid feeding pump may communicate with a first communication port that is provided at one end of the channel of the reaction processing vessel, and the inside of the pressurizing chamber may communicate with a second communication port that is provided at the other end of the channel of the reaction processing vessel. The reaction processor may further include a control unit that controls the liquid feeding pump in order to move the sample inside the channel.

The liquid feeding system may include a pressurizing chamber that has an internal pressure maintained to be higher than the air pressure in the surrounding environment of the reaction processor, preferably <NUM> atm or higher; a first liquid feeding pump that is arranged inside the pressurizing chamber; and a second liquid feeding pump that is arranged inside the pressurizing chamber. The output of the first liquid feeding pump may communicate with a first communication port that is provided at one end of the channel of the reaction processing vessel, and the output of the second liquid feeding pump may communicate with a second communication port that is provided at the other end of the channel of the reaction processing vessel. The reaction processor may further include a control unit that controls the first liquid feeding pump and the second liquid feeding pump in order to move the sample inside the channel.

The liquid feeding system may include: a pressurizing chamber that has an internal pressure maintained to be higher than the air pressure in the surrounding environment of the reaction processor, preferably <NUM> atm or higher; a liquid feeding chamber that has an internal pressure maintained to be a pressure that is higher than that inside the pressurizing chamber; a first direction switching valve that allows either one of the pressurizing chamber and the liquid feeding chamber to communicate with a first communication port provided at one end of the channel of the reaction processing vessel; and a second direction switching valve that allows either one of the pressurizing chamber and the liquid feeding chamber to communicate with a second communication port provided at the other end of the channel of the reaction processing vessel. The reaction processor may further include a control unit that controls the first direction switching valve and the second direction switching valve in order to move the sample inside the channel.

Another embodiment of the present invention relates to a reaction processing method. This method includes: placing a reaction processing vessel that is provided with a channel into which a sample is introduced; controlling the temperature of the channel in order to heat the sample inside the channel; and controlling the pressure inside the channel of the reaction processing vessel in order to move the sample inside the channel. The pressure inside the channel is maintained to be higher than the air pressure in the surrounding environment of the reaction processor, preferably <NUM> atm or higher, during a reaction process of the sample.

According to the present invention, a reaction processor, a reaction processing vessel, and a reaction processing method can be provided that are capable of performing PCR while preventing the boiling of a sample and the generation of air bubbles even in a place where the pressure is low.

An explanation will be given in the following regarding a reaction processor according to an embodiment of the present invention. This reaction processor is a device for performing PCR. The same or equivalent constituting elements, members, and processes illustrated in each drawing shall be denoted by the same reference numerals, and duplicative explanations will be omitted appropriately. Reference herein to details of the illustrated embodiments is not intended to limit the scope of the claims. It should be understood that not all of the features and the combination thereof discussed are essential to the invention.

<FIG> are diagrams for explaining a reaction processing vessel <NUM> usable in a reaction processor according to a first embodiment of the present invention. <FIG> is a plan view of the reaction processing vessel <NUM>, and <FIG> is a front view of the reaction processing vessel <NUM>.

As shown in <FIG>, the reaction processing vessel <NUM> comprises a substrate <NUM> and a channel sealing film <NUM>.

The substrate <NUM> is preferably formed of a material that is stable under temperature changes and is resistant to a sample solution that is used. Further, the substrate <NUM> is preferably formed of a material that has good moldability, a good transparency and barrier property, and a low self-fluorescence property. As such a material, an inorganic material such as glass, silicon (Si), or the like, a resin such as acrylic, polyester, silicone, or the like, and particularly cycloolefin are preferred. An example of the dimensions of the substrate <NUM> includes a long side of <NUM>, a short side of <NUM>, and a thickness of <NUM>.

A groove-like channel <NUM> is formed on the lower surface 14a of the substrate <NUM>, and this channel <NUM> is sealed by the channel sealing film <NUM>. The channel <NUM> is formed in a so-called serpiginous manner where a turn is continuously made by combining curved (turn) portions and straight portions in a plan view. Specifically, a combination of a pair of turn portions on respective sides (corresponding to a high temperature region and a low temperature region to be described later) and two straight portions (corresponding to a medium temperature region to be described later) connecting the pair of turn portions is defined as one unit, and units are formed in a continuous manner such that the number of the units is equal to or more than the scheduled number of thermal cycles to be applied to the sample. An example of the dimensions of the channel <NUM> formed on the lower surface 14a of the substrate <NUM> includes a width of <NUM> and a depth of <NUM>. A first communication port <NUM>, which communicates with the outside, is formed at the position of one end of the channel <NUM> in the substrate <NUM>. A second communication port <NUM> is formed at the position of the other end of the channel <NUM> in the substrate <NUM>. The pair, the first communication port <NUM> and the second communication port <NUM>, formed on the respective ends of the channel <NUM> is formed so as to be exposed on the upper surface 14b of the substrate <NUM>. Such a substrate can be produced by injection molding or cutting work with an NC processing machine or the like.

On the lower surface 14a of the substrate <NUM>, the channel sealing film <NUM> is attached. In the reaction processing vessel <NUM> according to the first embodiment, most of the channel <NUM> is formed in the shape of a groove exposed on the lower surface 14a of the substrate <NUM>. This is for allowing for easy molding by injection molding using a metal mold or the like. In order to seal this groove so as to make use of the groove as a channel, the channel sealing film <NUM> is attached on the lower surface 14a of the substrate <NUM>.

The channel sealing film <NUM> may be sticky on one of the main surfaces thereof or may have a functional layer that exhibits stickiness or adhesiveness by pressing that is formed on one of the main surfaces. Thus, the channel sealing film <NUM> has a function of being easily able to become integral with the lower surface 14a of the substrate <NUM> while being in close contact with the lower surface 14a. The channel sealing film <NUM> is desirably formed of a material, including an adhesive, that has a low self-fluorescence property. In this respect, a transparent film made of a resin such as a cycloolefin polymer, polyester, polypropylene, polyethylene or acrylic is suitable but is not limited thereto. Further, the channel sealing film <NUM> maybe formed of a plate-like glass or resin. Since rigidity can be expected in this case, the channel sealing film <NUM> is useful for preventing warpage and deformation of the reaction processing vessel <NUM>.

<FIG> schematically shows a state where a sample <NUM> is introduced into the channel <NUM> of the reaction processing vessel <NUM>. In <FIG>, in order to emphasize the position of the sample <NUM>, the sample <NUM> is shown by a solid line that is thicker than that for the channel <NUM>. It should be noted that the solid line does not indicate a state where the sample <NUM> overflows outside the channel.

The sample <NUM> is introduced into the channel <NUM> through either one of the first communication port <NUM> and the second communication port <NUM>. The method for the introduction is not limited to this. Alternatively, for example, an appropriate amount of sample may be directly introduced through the communication port using a pipette, a dropper, a syringe, or the like. Alternatively, a method of introduction may be used that is performed while preventing contamination via a cone-shaped needle chip, in which a filter made of porous PTFE or polyethylene is incorporated. In general, many types of such needle chips are sold and can be obtained easily, and the needle chips can be used while being attached to the tip of a pipette, a dropper, a syringe, or the like. Furthermore, the sample may be moved to a predetermined position in the channel as shown in <FIG> by discharging and introducing the sample by a pipette, a dropper, a syringe, or the like and then further pushing the sample through pressurization. Regarding the so-called initial position of the sample, as shown in <FIG>, an example is shown where a position in the high temperature region <NUM> described later is set as the initial position. However, the initial position is not limited thereto.

The sample <NUM> includes, for example, those obtained by adding a plurality of types of primers, a thermostable enzyme and four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP) as PCR reagents to a mixture containing two or more types of DNA. Further, a fluorescent probe that specifically reacts to DNA subjected to a reaction process is mixed. Commercially available real-time PCR reagent kits and the like can be also used.

<FIG> is a schematic diagram for explaining a reaction processor <NUM> according to the first embodiment of the present invention. <FIG> is a diagram for explaining a state where the reaction processing vessel <NUM> is set at a predetermined position of the reaction processor <NUM>.

The reaction processor <NUM> according to the first embodiment is provided with a reaction processing vessel placing portion (not shown) on which the reaction processing vessel <NUM> is placed, a temperature control system <NUM>, and a CPU <NUM>. As shown in <FIG>, the temperature control system <NUM> is configured so as to be able to accurately maintain and control the temperature of a region <NUM>, which is approximately the lower one third of the figure page in the channel <NUM> of the reaction processing vessel <NUM> placed on the reaction processing vessel placing portion, the temperature of a region <NUM>, which is approximately the upper one third of the figure page, and the temperature of a region <NUM>, which is approximately the middle one third of the figure page, to be three levels of temperature of about <NUM>, about <NUM>, and about <NUM>, respectively. Hereinafter, the region <NUM> of the channel <NUM> is referred to as "high temperature region <NUM>", the region <NUM> of the channel <NUM> is referred to as "medium temperature region <NUM>", and the region <NUM> of the channel <NUM> is referred to as "low temperature region <NUM>", and the regions are collectively referred to as a thermal cycle region.

The temperature control system <NUM> is for maintaining the temperature of each temperature region of the thermal cycle region and is specifically provided with a high temperature heater <NUM> for heating the high temperature region <NUM> of the channel <NUM>, a medium temperature heater <NUM> for heating the medium temperature region <NUM> of the channel <NUM>, a low temperature heater <NUM> for heating the low temperature region <NUM> of the channel <NUM>, a temperature sensor (not shown) such as, for example, a thermocouple or the like for measuring the actual temperature of each temperature region, a high temperature heater driver <NUM> for controlling the temperature of the high temperature heater <NUM>, a medium temperature heater driver <NUM> for controlling the temperature of the medium temperature heater <NUM>, a low temperature heater driver <NUM> for controlling the temperature of the low temperature heater <NUM>. Information on the actual temperature measured by the temperature sensor is sent to the CPU <NUM>. Based on the information on the actual temperature of each temperature region, the CPU <NUM> controls each heater driver such that the temperature of each heater becomes a predetermined temperature. Each heater may be, for example, a resistance heating element, a Peltier element, or the like. The temperature control system <NUM> may be further provided with other components for improving the temperature controllability of each temperature region.

The reaction processor <NUM> according to the first embodiment is further provided with a liquid feeding system <NUM> for moving the sample <NUM> inside the channel <NUM> of the reaction processing vessel <NUM>. By controlling the pressure inside the channel <NUM> using this liquid feeding system <NUM>, the sample <NUM> is continuously moved in one direction inside the channel <NUM> such that the sample <NUM> can pass through each temperature region inside the thermal cycle region of the reaction processing vessel <NUM>, and, as a result, a thermal cycle can be applied to the sample <NUM>. More specifically, target DNA in the sample <NUM> is selectively amplified by applying a step of denaturation in the high temperature region <NUM>, a step of annealing in the low temperature region <NUM>, and a step of elongation in the medium temperature region <NUM>. In other words, the high temperature region <NUM> can be considered to be a denaturation temperature region, the low temperature region <NUM> can be considered to be an annealing temperature region, and the medium temperature region <NUM> can be considered to be an elongation temperature region. The period of time for staying in each temperature region can be appropriately set by changing the period of time during which the sample stops at a predetermined position in each temperature region, the speed at which the sample moves, the size (area) of each temperature region, a channel length corresponding to each temperature region, and the like. Further, the annealing temperature region and the elongation temperature region may be combined into an annealing and elongation temperature region. In this case, the thermal cycle region is formed of temperature regions of two levels: a high temperature region for denaturation; and a temperature region (medium-low temperature region) where the temperature is lower than that of the high temperature region.

The liquid feeding system <NUM> is provided with a pressurizing chamber <NUM>, a liquid feeding pump <NUM>, a liquid feeding pump driver <NUM> for controlling the liquid feeding pump <NUM>, a pressurizing chamber pump <NUM>, a pressurizing chamber pump driver <NUM> for controlling the pressurizing chamber pump <NUM>, a first tube <NUM>, and a second tube <NUM>.

A first end portion 46a of the first tube <NUM> is connected to the first communication port <NUM> of the reaction processing vessel <NUM>. A packing material or a seal for securing airtightness is preferably arranged at the connection between the first communication port <NUM> and the first end portion 46a of the first tube <NUM>. A second end portion 46b of the first tube <NUM> is connected to the output of the liquid feeding pump <NUM>. The liquid feeding pump <NUM> may be, for example, a micro blower pump comprising a diaphragm pump.

The CPU <NUM> controls the air supply and pressurization from the liquid feeding pump <NUM> via the liquid feeding pump driver <NUM>. The air supply and pressurization from the liquid feeding pump <NUM> act on the sample <NUM> inside the channel <NUM> through the first communication port <NUM> and becomes a propulsive force to move the sample <NUM>.

As the liquid feeding pump <NUM>, for example, a micro blower pump (MZB1001 T02 model) manufactured by Murata Manufacturing Co. , or the like can be used. While this micro blower pump can increase the pressure on a secondary side to be higher than a primary side during operation, the pressure on the primary side and the pressure on the secondary side become equal at the moment when the pump is stopped or when the pump is stopped. In the first embodiment, the liquid feeding pump <NUM> is entirely arranged inside the pressurizing chamber <NUM>.

A first end portion 47a of the second tube <NUM> is connected to the second communication port <NUM> of the reaction processing vessel <NUM>. A packing material or a seal for securing airtightness is preferably arranged at the connection between the second communication port <NUM> and the first end portion 47a of the second tube <NUM>. A second end portion 47b of the second tube <NUM> is connected so as to communicate with the inside of the pressurizing chamber <NUM>. As a result, the second communication port <NUM> of the reaction processing vessel <NUM> communicates with the atmosphere inside the pressurizing chamber <NUM>.

The pressurizing chamber <NUM> forms a space having a certain volume therein. The pressurizing chamber pump <NUM> is connected to the pressurizing chamber <NUM>. The pressurizing chamber pump driver <NUM> controls the pressurizing chamber pump <NUM> such that the space inside the pressurizing chamber <NUM> has a predetermined pressure in accordance with an instruction from the CPU36. As the pressurizing chamber pump <NUM>, for example, a compact DC diaphragm pump (DSA-<NUM>-12BL model) manufactured by Denso Sangyo Co. , or the like can be used, and a means of pressurization by a rubber ball, a syringe, or the like can be also used as a simple means. In the first embodiment, the pressure inside the pressurizing chamber <NUM> is set to be higher than the air pressure in the surrounding environment of the reaction processor <NUM> during the reaction process and more preferably maintained at <NUM> atm (<NUM> hPa) or higher. The air pressure in the surrounding environment of the reaction processor means the pressure (or atmospheric pressure) at a place where the reaction processor according to the present invention is installed, a place where the reaction process is performed by the processor, or, when the reaction processor is installed at a place that is partitioned from the surroundings, the partitioned place. The pressure inside the pressurizing chamber <NUM> needs to be applied to such an extent that significant evaporation of the sample and generation of air bubbles or the like, which affect a PCR reaction process, can be prevented even when the sample is repeatedly exposed to a high temperature (about <NUM>). The higher the pressure inside the pressurizing chamber <NUM> becomes, the more the influence of the evaporation of the sample and the like can be suppressed. However, on the other hand, the liquid feeding system <NUM> becomes complicated or enlarged including the handling thereof. Thus, a person skilled in the art can comprehensively judge the application, purpose, cost, effect, etc., of the processor so as to design the entire system.

An atmospheric pressure releasing valve <NUM> is provided in the pressurizing chamber <NUM>. The atmospheric pressure releasing valve <NUM> is controlled such that the pressure of the liquid feeding system <NUM> (the inside of the pressurizing chamber <NUM>, the first tube <NUM>, the second tube <NUM>, etc.) and the pressure in the channel <NUM> of the reaction processing vessel <NUM> become equal to the air pressure in the surrounding environment of the reaction processor <NUM> at the time of installing or removing the reaction processing <NUM> in or from the reaction processor <NUM>. Thereby, rapid movement and squirting of the sample <NUM> can be prevented.

Further, a pressure sensor (not shown) for constantly monitoring the pressure of the internal space thereof may be provided in the pressurizing chamber <NUM>. By sending the actual pressure detected by the pressure sensor to the CPU <NUM>, the pressure inside the pressurizing chamber <NUM> can be suitably controlled.

The reaction processor <NUM> according to the first embodiment is further provided with a fluorescence detector <NUM>. Fluorescence from the sample <NUM> in the channel <NUM> of the reaction processing vessel <NUM> can be detected using the fluorescence detector <NUM>, and the value thereof can be used as an index serving as information for determining the progress of the PCR or the termination of the reaction.

As the fluorescence detector <NUM>, an optical fiber-type fluorescence detector FLE-<NUM> manufactured by Nippon Sheet Glass Co. , can be used, which is a very compact optical system that allows for rapid measurement and the detection of fluorescence regardless of whether the place is a lighted place or a dark place. This optical fiber-type fluorescence detector allows the wavelength characteristic of the excitation light/fluorescence to be tuned such that the wavelength characteristic is suitable for the characteristic of fluorescence emitted from the sample <NUM> and thus allows an optimum optical and detection system for a sample having various characteristics to be provided.

The optical fiber-type fluorescence detector <NUM> is provided with an optical head <NUM>, a fluorescence detector driver <NUM>, and an optical fiber <NUM> connecting the optical head <NUM> and the fluorescence detector driver <NUM>. The fluorescence detector driver <NUM> includes a light source for excitation light (LED, laser or other light sources adjusted to emit specific wavelengths), an optical fiber-type multiplexer/demultiplexer and a photoelectric conversion device (PD, APD, or a light detector such as a photomultiplier) (neither of which is shown), and a driver or the like for controlling these. The optical head <NUM> is formed of an optical system such as a lens and has a function of directionally irradiating the sample with excitation light and collecting fluorescence emitted from the sample. The collected fluorescence is separated from the excitation light by the optical fiber-type multiplexer/demultiplexer inside the fluorescence detector driver <NUM> through the optical fiber <NUM> and converted into an electric signal by the photoelectric conversion element. For example, as shown in <FIG>, the optical head <NUM> may be arranged near the second communication port <NUM> of the reaction processing vessel <NUM>. In this case, the completion of the amplification of DNA can be learned by detecting the fluorescence from the sample <NUM> sent near the second communication port <NUM> after the completion of the series of reaction processes. Further, a plurality of optical heads <NUM> may be arranged so as to be able to detect fluorescence from the sample <NUM> near the first communication port <NUM> or in the channel <NUM> along the way. By monitoring a change in the fluorescence signal along the channel <NUM>, the amplification of the DNA can be known in a time series manner. The fluorescence detector is not limited to an optical fiber-type fluorescence detector as long as the fluorescence detector exhibits the function of detecting fluorescence from a sample.

A description will be given of a reaction processing method in which the reaction processor <NUM> configured as described above is used. In the initial state of the processor, it is assumed that the second end portion 46b of the first tube <NUM> is connected to the output of the liquid feeding pump <NUM> and that the first end portion 46a of the first tube <NUM> is open. Further, it is assumed that the second end portion 47b of the second tube <NUM> is connected to the pressurizing chamber <NUM> and that the first end portion 47a of the second tube <NUM> is open.

First, the sample <NUM> is introduced into the reaction processing vessel <NUM> and moved to the initial position, and then the reaction processing vessel <NUM> is set on the reaction processing vessel placing portion of the reaction processor <NUM>.

Next, the atmospheric pressure releasing valve <NUM> provided in the pressurizing chamber <NUM> is opened such that the respective pressures in the pressurizing chamber <NUM> and in the first tube <NUM> and the second tube <NUM> to be connected respectively to the first communication port <NUM> and the second communication port <NUM> of the reaction processing vessel <NUM> become equal to the atmospheric pressure. Subsequently, the first end portion 46a of the first tube <NUM> extending from the liquid feeding pump <NUM> is connected to the first communication port <NUM> of the reaction processing vessel <NUM>, and the first end portion 47a of the second tube <NUM> extending from the pressurizing chamber <NUM> is connected to the second communication port <NUM> of the reaction processing vessel <NUM>. Neither the liquid feeding pump <NUM> nor the pressurizing chamber pump <NUM> is operated at this point. Subsequently, the atmospheric pressure releasing valve <NUM> provided in the pressurizing chamber <NUM> is closed.

Next, the pressurizing chamber pump <NUM> is operated such that the pressure inside the pressurizing chamber <NUM> and in the channel <NUM> of the reaction processing vessel <NUM> communicating with the pressurizing chamber <NUM> is higher than the air pressure in the surrounding environment of the reaction processor <NUM>, preferably <NUM> atm (<NUM> hPa) or higher. Since the liquid feeding pump <NUM> is not operated at this time, the pressure on the primary side and the pressure on the secondary side are equal, that is, the pressure of the first communication port <NUM> communicating with the secondary side of the liquid feeding pump <NUM> is also equal to the pressure inside the pressurizing chamber <NUM>. Therefore, since the pressures in the spaces on respective sides (the first communication port <NUM> side and the second communication port <NUM> side) of the sample <NUM> in the channel <NUM> of the reaction processing vessel <NUM> are equal, the sample <NUM> does not move. Since the pressure in the sample <NUM> and the pressure inside the channel <NUM> including the sample <NUM> are higher than the air pressure in the surrounding environment of the reaction processor <NUM> and are preferably <NUM> atm or higher, even under a low atmospheric pressure environment such as a high altitude place, the boiling and foaming of the sample <NUM> caused due to the lowering of the boiling point of the sample <NUM> mainly composed of an aqueous solution can be prevented.

Subsequently, the temperature control system <NUM> is operated so as to start the temperature control of each temperature region in the reaction processing vessel <NUM>. The temperature control may be put on hold for a predetermined amount of time until the temperature in each temperature region is stabilized. The temperature control is preferably started after the pressure inside the channel <NUM> is stabilized by the liquid feeding system <NUM>.

Next, the liquid feeding pump <NUM> is operated by the liquid feeding pump driver <NUM>. Thereby, the pressure inside the channel <NUM> on the first communication port <NUM> side becomes higher than that on the second communication port <NUM> side in the channel <NUM> on both sides of the sample <NUM>, and the sample <NUM> can thus move toward the second communication port <NUM> while being pushed inside the channel <NUM>. The sample <NUM> cyclically and continuously passes through each temperature region of the denaturation region (high temperature region <NUM>), the annealing region (low temperature region <NUM>), and the elongation region (medium temperature region <NUM>) along the continuous serpentine channel <NUM>. Further, in the case of a reaction processor in which temperature regions of two levels are set, the sample <NUM> cyclically and continuously passes through each temperature region of the denaturation region (high temperature region) and the annealing and elongation region (medium-low temperature region). This allows a predetermined number of thermal cycles to be applied to the sample <NUM> and allows PCR to occur such that predetermined DNA can be selectively amplified.

As described above, in the reaction processor <NUM> according to the first embodiment, the pressure inside the channel <NUM> of the reaction processing vessel <NUM> is always maintained to be higher than the air pressure in the surrounding environment of the reaction processor <NUM>, preferably <NUM> atm or higher, during the reaction process. In other words, during the reaction process, the sample <NUM> is constantly pressurized to have a pressure higher than the air pressure in the surrounding environment of the reaction processor <NUM>, preferably <NUM> atm or higher. Therefore, PCR can be performed while preventing the boiling of a sample and the generation of air bubbles even in a place where the air pressure is low such as a high altitude place or the inside of an airplane.

<FIG> are diagrams for explaining a reaction processing vessel <NUM> usable in a reaction processor according to a second embodiment of the present invention. <FIG> is a plan view of the reaction processing vessel <NUM>, and <FIG> is a front view of the reaction processing vessel <NUM>.

As shown in <FIG>, the reaction processing vessel <NUM> comprises a substrate <NUM> and a channel sealing film <NUM>. The respective structures such as materials, dimensions, and the like of the substrate <NUM> and the channel sealing film <NUM> are the same as those of the reaction processing vessel <NUM> explained in the first embodiment. A groove-like channel <NUM> is formed on the lower surface 114a of the substrate <NUM>, and this channel <NUM> is sealed by the channel sealing film <NUM>. An example of the dimensions of the channel <NUM> formed on the lower surface 114a of the substrate <NUM> includes a width of <NUM> and a depth of <NUM>. A first communication port <NUM>, which communicates with the outside, is formed at the position of one end of the channel <NUM> in the substrate <NUM>. A second communication port <NUM> is formed at the position of the other end of the channel <NUM> in the substrate <NUM>. The pair, the first communication port <NUM> and the second communication port <NUM>, formed on the respective ends of the channel <NUM> is formed so as to be exposed on the upper surface 114b of the substrate <NUM>. On the lower surface 114a of the substrate <NUM>, the channel sealing film <NUM> is attached. In the reaction processing vessel <NUM> according to the second embodiment, most of the channel <NUM> is formed in the shape of a groove exposed on the lower surface 114a of the substrate <NUM>. This is for allowing for easy molding by injection molding using a metal mold or the like or by cutting work by an NC processing machine. In order to seal this groove so as to make use of the groove as a channel, the channel sealing film <NUM> is attached on the lower surface 114a of the substrate <NUM>.

The reaction processing vessel <NUM> according to the second embodiment is provided with a temperature range, in which the control of temperatures of a plurality of levels is possible, in the channel <NUM> between a pair of communication ports just like the reaction processing vessel <NUM> according to the first embodiment. However, the reaction processing vessel <NUM> is different from the reaction processing vessel <NUM> according to the first embodiment in that the channel <NUM> is not a serpentine channel that is folded back in a continuous manner. As will be described later, this is because a sample is intended to be sent so as to continuously reciprocate between temperature regions, where temperatures of a plurality of levels are maintained, of at least one channel <NUM> instead of sending a sample in a one-way continuous flow to a serpentine channel that is folded back in a continuous manner as in the reaction processing vessel <NUM> according to the first embodiment. A portion of the channel <NUM> that corresponds to each temperature region in the channel <NUM> may have a serpentine shape (smaller compared to that according to the first embodiment) formed of a curved (turn) portion and a straight portion in the temperature region, and the temperature regions are connected by, for example, a short channel. Since the area and channel length of each temperature region can be made smaller than those of the first embodiment, it is relatively easy to reduce variations in temperature in each temperature region, and there is also an advantage that the entire channel length can be shortened such that the reaction processing vessel and the reaction processor can be made small.

<FIG> is a schematic diagram for explaining a reaction processor <NUM> according to the second embodiment of the present invention. <FIG> is a diagram for explaining a state where the reaction processing vessel <NUM> is set at a predetermined position of the reaction processor <NUM>. In <FIG>, a sample <NUM> is introduced into the channel <NUM> of the reaction processing vessel <NUM>. In <FIG>, in the same way as in the first embodiment, in order to emphasize the position of the sample <NUM>, the sample <NUM> is shown by a solid line that is thicker than that for the channel <NUM>. It should be noted that the solid line does not indicate a state where the sample <NUM> overflows outside the channel. In the second embodiment, the sample <NUM>, the method of introducing the sample <NUM>, etc., are also the same as those in the first embodiment. Regarding the initial position of the sample <NUM>, as shown in <FIG>, an example is shown where a position in the high temperature region <NUM> described later is set as the initial position. However, the initial position is not limited thereto.

The reaction processor <NUM> according to the second embodiment is provided with a reaction processing vessel placing portion (not shown) on which the reaction processing vessel <NUM> is placed, a temperature control system <NUM>, and a CPU <NUM>. As shown in <FIG>, the temperature control system <NUM> is configured so as to be able to accurately maintain and control the temperature of a region <NUM>, which is approximately the right one third of the figure page in the channel <NUM> of the reaction processing vessel <NUM> placed on the reaction processing vessel placing portion, the temperature of a region <NUM>, which is approximately the left one third of the figure page, and the temperature of a region <NUM>, which is approximately the middle one third of the figure page, to be three levels of temperature of about <NUM>, about <NUM>, and about <NUM>, respectively. Hereinafter, the region <NUM> of the channel <NUM> is referred to as "high temperature region <NUM>", the region <NUM> of the channel <NUM> is referred to as "medium temperature region <NUM>", and the region <NUM> of the channel <NUM> is referred to as "low temperature region <NUM>", and the regions are collectively referred to as a thermal cycle region.

The temperature control system <NUM> is for maintaining each temperature region of the thermal cycle region and is specifically provided with a high temperature heater <NUM> for heating the high temperature region <NUM> of the channel <NUM>, a medium temperature heater <NUM> for heating the medium temperature region <NUM> of the channel <NUM>, a low temperature heater <NUM> for heating the low temperature region <NUM> of the channel <NUM>, a temperature sensor (not shown) such as, for example, a thermocouple or the like for measuring the actual temperature of each temperature region, a high temperature heater driver <NUM> for controlling the temperature of the high temperature heater <NUM>, a medium temperature heater driver <NUM> for controlling the temperature of the medium temperature heater <NUM>, a low temperature heater driver <NUM> for controlling the temperature of the low temperature heater <NUM>. Information on the actual temperature measured by the temperature sensor is sent to the CPU <NUM>. Based on the information on the actual temperature of each temperature region, the CPU <NUM> controls each heater driver such that the temperature of each heater becomes a predetermined temperature. Each heater may be, for example, a resistance heating element, a Peltier element, or the like. The temperature control system <NUM> may be further provided with other components for improving the temperature controllability of each temperature region.

The reaction processor <NUM> according to the second embodiment is further provided with a liquid feeding system <NUM> for moving the sample <NUM> inside the channel <NUM> of the reaction processing vessel <NUM>. By controlling the pressure inside the channel <NUM> using this liquid feeding system <NUM>, the sample <NUM> is continuously moved inside the channel <NUM> in a reciprocating manner such that the sample <NUM> can pass through each temperature region inside the thermal cycle region of the reaction processing vessel <NUM>, and, as a result, a thermal cycle can be applied to the sample <NUM>. More specifically, target DNA in the sample <NUM> is selectively amplified by applying a step of denaturation in the high temperature region <NUM>, a step of annealing in the low temperature region <NUM>, and a step of elongation in the medium temperature region <NUM>. In other words, the high temperature region <NUM> can be considered to be a denaturation temperature region, the low temperature region <NUM> can be considered to be an annealing temperature region, and the medium temperature region <NUM> can be considered to be an elongation temperature region. The period of time for staying in each temperature region can be appropriately set by changing the period of time during which the sample stops at a predetermined position in each temperature region, the speed at which the sample moves, the size (area) of each temperature region, a channel length corresponding to each temperature region, and the like. Further, the annealing temperature region and the elongation temperature region may be combined into an annealing and elongation temperature region. In this case, the thermal cycle region is formed of temperature regions of two levels: a high temperature region for denaturation; and a temperature region (medium-low temperature region) where the temperature is lower than that of the high temperature region.

The liquid feeding system <NUM> is provided with a pressurizing chamber <NUM>, a first liquid feeding pump <NUM>, a first liquid feeding pump driver <NUM> for controlling the first liquid feeding pump <NUM>, a second liquid feeding pump <NUM>, a second liquid feeding pump driver <NUM> for controlling the second liquid feeding pump <NUM>, a pressurizing chamber pump <NUM>, a pressurizing chamber pump driver <NUM> for controlling the pressurizing chamber pump <NUM>, a first tube <NUM>, and a second tube <NUM>.

A first end portion 146a of the first tube <NUM> is connected to the first communication port <NUM> of the reaction processing vessel <NUM>. A packing material or a seal for securing airtightness is preferably arranged at the connection between the first communication port <NUM> and the first end portion 146a of the first tube <NUM>. A second end portion 146b of the first tube <NUM> is connected to the output of the first liquid feeding pump <NUM>. The first liquid feeding pump <NUM> may be, for example, a micro blower pump comprising a diaphragm pump. In the same way, a first end portion 147a of the second tube <NUM> is connected to the second communication port <NUM> of the reaction processing vessel <NUM>. A packing material or a seal for securing airtightness is preferably arranged at the connection between the second communication port <NUM> and the first end portion 147a of the second tube <NUM>. A second end portion 147b of the second tube <NUM> is connected to the output of the second liquid feeding pump <NUM>. The second liquid feeding pump <NUM> may be, for example, a micro blower pump comprising a diaphragm pump.

The CPU <NUM> controls the air supply and pressurization from the first liquid feeding pump <NUM> and the second liquid feeding pump <NUM> via the first liquid feeding pump driver <NUM> and the second liquid feeding pump driver <NUM>. The air supply and pressurization from the first liquid feeding pump <NUM> and the second liquid feeding pump <NUM> act on the sample <NUM> inside the channel <NUM> through the first communication port <NUM> and the second communication port <NUM> and becomes a propulsive force to move the sample <NUM>.

As the first liquid feeding pump <NUM> and the second liquid feeding pump <NUM>, for example, a micro blower pump (MZB1001 T02 model) manufactured by Murata Manufacturing Co. , or the like can be used. In the second embodiment, the first liquid feeding pump <NUM> and the second liquid feeding pump <NUM> are both entirely arranged inside the pressurizing chamber <NUM>.

The pressurizing chamber <NUM> forms a space having a certain volume therein. A pressurizing chamber pump <NUM> is connected to the pressurizing chamber <NUM>. The pressurizing chamber pump driver <NUM> controls the pressurizing chamber pump <NUM> such that the space inside the pressurizing chamber <NUM> has a predetermined pressure in accordance with an instruction from the CPU <NUM>. As the pressurizing chamber pump <NUM>, a compact DC diaphragm pump (DSA-<NUM>-12BL model) manufactured by Denso Sangyo Co. , or the like can be used, and a means of pressurization by a rubber ball, a syringe, or the like can be also used as a simple means. In the second embodiment, the pressure inside the pressurizing chamber <NUM> is set to be higher than the air pressure in the surrounding environment of the reaction processor <NUM> during the reaction process and more preferably maintained at <NUM> atm (<NUM> hPa) or higher. The pressure inside the pressurizing chamber <NUM> needs to be applied to such an extent that significant evaporation of the sample and generation of air bubbles or the like, which affect the PCR reaction process, can be prevented even when the sample is repeatedly exposed to a high temperature (about <NUM>). The higher the pressure inside the pressurizing chamber <NUM> becomes, the more the influence of the evaporation of the sample and the like can be suppressed. However, on the other hand, the liquid feeding system <NUM> becomes complicated or enlarged including the handling thereof. Thus, a person skilled in the art can comprehensively judge the application, purpose, cost, effect, etc., of the processor so as to design the entire system.

An atmospheric pressure releasing valve <NUM> is provided in the pressurizing chamber <NUM>. The atmospheric pressure releasing valve <NUM> is controlled such that the pressure of the liquid feeding system <NUM> and the pressure of the reaction processing vessel <NUM> in the channel <NUM> become equal to the atmospheric pressure at the time of installing or removing the reaction processing vessel <NUM>. Thereby, rapid movement and squirting of the sample <NUM> can be prevented.

The reaction processor <NUM> according to the second embodiment is further provided with a fluorescence detector <NUM>. Fluorescence from the sample <NUM> in the channel <NUM> of the reaction processing vessel <NUM> can be detected using the fluorescence detector <NUM>, and the value thereof can be used as an index serving as information for determining the progress of the PCR or the termination of the reaction.

As the fluorescence detector <NUM>, an optical fiber-type fluorescence detector can be used in the same way as in the first embodiment. The optical fiber-type fluorescence detector <NUM> is provided with a first optical head <NUM>, a second optical head <NUM>, a first fluorescence detector driver <NUM>, a second fluorescence detector driver <NUM>, a first optical fiber <NUM> connecting the first optical head <NUM> and the first fluorescence detector driver <NUM>, and a second optical fiber <NUM> connecting the second optical head <NUM> and the second fluorescence detector driver <NUM>. The combination of the first optical head <NUM>, the first fluorescence detector driver <NUM>, and the first optical fiber <NUM> can be also referred to as a first fluorescence detector, and the combination of the second optical head <NUM>, the second fluorescence detector driver <NUM>, and the second optical fiber <NUM> can be also referred to as a second fluorescence detector. Furthermore, a third fluorescence detector, a fourth fluorescence detector, and a higher-order fluorescence detector maybe provided. For the first and second fluorescence detectors, those having the same structures as those according to the first embodiment can be used, respectively, and the detailed description thereof will be thus omitted. Further, the first and second fluorescence detectors may have the same characteristics (for example, the target wavelengths of excitation light and fluorescence are the same) or may have different characteristics (such as different target wavelengths). In this case, it is advantageous in that amplification of a plurality of types of DNA having different fluorescence characteristics can be known in some cases.

As shown in <FIG>, the first optical head <NUM> is arranged in a channel connecting the high temperature region <NUM> and the medium temperature region <NUM>. The second optical head <NUM> is arranged in a channel connecting the medium temperature region <NUM> and the low temperature region <NUM>. Since the reaction progresses while the sample <NUM> is fed in a reciprocating manner in the channel <NUM> and predetermined DNA contained in the sample <NUM> is amplified, by monitoring a change in the amount of fluorescence obtained from the sample, the progress of the DNA amplification can be learned in real time. In a serpentine channel having a continuous flow moving in one direction according to the first embodiment, it is substantially difficult to check the progress of DNA amplification in real time. This is because it is necessary to appropriately install far more fluorescent detectors on a long channel compared to the number of fluorescent detectors according to the second embodiment and scan along the channel of the fluorescence detectors. The reaction processing vessel comprising a serpentine channel having a reciprocating flow according to the second embodiment is also advantageous in this point.

A description will be given of a reaction processing method in which the reaction processor <NUM> configured as described above is used. In the initial state of the processor, it is assumed that the second end portion 146b of the first tube <NUM> is connected to the output of the liquid feeding pump <NUM> and that the first end portion 146a of the first tube <NUM> is open. Also, it is assumed that the second end portion 147b of the second tube <NUM> is connected to the output of the second liquid feeding pump <NUM> and that the first end portion 147a of the second tube <NUM> is open.

Next, the atmospheric pressure releasing valve <NUM> provided in the pressurizing chamber <NUM> is opened such that the respective pressures in the pressurizing chamber <NUM> and in the first tube <NUM> and the second tube <NUM> to be connected respectively to the first communication port <NUM> and the second communication port <NUM> of the reaction processing vessel <NUM> become equal to the atmospheric pressure. Subsequently, the first end portion 146a of the first tube <NUM> extending from the first liquid feeding pump <NUM> is connected to the first communication port <NUM> of the reaction processing vessel <NUM>, and the first end portion 147a of the second tube <NUM> extending from the second liquid feeding pump <NUM> is connected to the second communication port <NUM> of the reaction processing vessel <NUM>. None of the first liquid feeding pump <NUM>, the second liquid feeding pump <NUM>, and the pressurizing chamber pump <NUM> is operated at this point. Subsequently, the atmospheric pressure releasing valve <NUM> provided in the pressurizing chamber <NUM> is closed.

Next, the pressurizing chamber pump <NUM> is operated by the pressurizing chamber pump driver <NUM> such that the pressure inside the pressurizing chamber <NUM> and in the channel <NUM> of the reaction processing vessel <NUM> communicating with the pressurizing chamber <NUM> is higher than the air pressure in the surrounding environment of the reaction processor <NUM>, preferably <NUM> atm (<NUM> hPa) or higher. Since neither the first liquid feeding pump <NUM> nor the second liquid feeding pump <NUM> is in operation at this time, the pressure on the primary side and the pressure on the secondary side are equal, that is, the pressure of the first communication port <NUM> on the secondary side and the pressure of the second communication port <NUM> are also equal to the pressure inside the pressurizing chamber <NUM>. Therefore, since the pressures in the spaces on respective sides (the first communication port <NUM> side and the second communication port <NUM> side) of the sample <NUM> in the channel <NUM> of the reaction processing vessel <NUM> are equal, the sample <NUM> does not move. Since the pressure in the sample <NUM> and the pressure inside the channel <NUM> including the sample <NUM> are always higher than the air pressure in the surrounding environment of the reaction processor <NUM> and are preferably <NUM> atm or higher, even under a low atmospheric pressure environment such as a high altitude place, the boiling and foaming of the sample <NUM> caused due to the lowering of the boiling point of the sample <NUM> mainly composed of an aqueous solution can be prevented.

Subsequently, the temperature control system <NUM> is operated so as to start the temperature control of each temperature region in the reaction processing vessel <NUM>. The temperature control may be put on hold for a predetermined amount of time until the temperature in each temperature region is stabilized. The temperature control is preferably started after the pressure inside the channel <NUM> is kept to be a certain pressure or higher by the liquid feeding system <NUM>.

It is assumed that the initial position of the sample <NUM> is located, for example, in the high temperature region <NUM>. When the sample <NUM> is in the high temperature region <NUM> for a certain period of time, denaturation of the DNA occurs. First, the first liquid feeding pump <NUM> is operated. Thereby, the pressure inside the channel <NUM> on the first communication port <NUM> side becomes higher than that on the second communication port <NUM> side in the spaces on both sides of the sample <NUM>, and the sample <NUM> can thus move from the high temperature region <NUM> to the low temperature region <NUM> via the medium temperature region <NUM> while being pushed inside the channel <NUM> toward the second communication port <NUM>. When the sample <NUM> reaches the low temperature region <NUM>, the first liquid feeding pump <NUM> is stopped. When the first liquid feeding pump <NUM> is stopped, the pressure on the primary side and the pressure on the secondary side become equal as described above. Thus, the pressure in the space on the first communication port <NUM> side of the sample <NUM> and the pressure in the channel space on the second communication port <NUM> side of the sample <NUM> both become equal to the pressure inside the pressurizing chamber <NUM> (i.e., there is no difference), and the sample <NUM> thus stops moving. Placing the sample <NUM> in the low temperature region <NUM> for a certain period of time causes annealing of the DNA.

Subsequently, the second liquid feeding pump <NUM> is operated, and, when the movement of the sample <NUM> from the low temperature region <NUM> to the medium temperature region <NUM> is completed, the second liquid feeding pump <NUM> is stopped. Placing the sample <NUM> in the medium temperature region <NUM> for a certain period of time causes elongation of the DNA. Further, the second liquid feeding pump <NUM> is operated, and, when the movement of the sample <NUM> from the medium temperature region <NUM> to the high temperature region <NUM> is completed, the second liquid feeding pump <NUM> is stopped. Placing the sample <NUM> in the high temperature region <NUM> for a certain period of time causes denaturation of the DNA.

By controlling the operation of the liquid feeding system <NUM> so as to repeat the movement of the sample <NUM> described above, the sample <NUM> reciprocates inside the channel <NUM>. More specifically, the sample <NUM> cyclically passes through the respective regions of the temperatures: a high temperature (denaturation); a low temperature (annealing); a medium temperature (elongation); a high temperature (denaturation); a low temperature (annealing); a medium temperature (elongation); and so on. Further, in the case of a reaction processor where temperature regions of two levels are set, the sample <NUM> cyclically passes through the respective regions of the temperatures: a high temperature (denaturation); a medium-low temperature (annealing and elongation) ; a high temperature (denaturation); a medium-low temperature (annealing and elongation); and so on. This allows a predetermined number of thermal cycles to be applied to the sample <NUM> and allows PCR to occur such that predetermined DNA can be selectively amplified.

In the reaction processor <NUM> according to the second embodiment, since the sample <NUM> continuously reciprocates inside a single channel <NUM> connecting a plurality of temperature regions, it is important to control the position of the sample <NUM>. Therefore, the fluorescence detector <NUM> described above can be allowed to function as a position sensor. If the optical head of the fluorescence detector <NUM> is arranged so as to detect fluorescence emitted from the sample <NUM> at a specific location in the channel <NUM>, a fluorescence signal is at zero or at a background level when the sample <NUM> is not at the specific location, and the fluorescence signal exhibits a change in output rising from zero or the background level and then going back to zero or the background level again when the sample <NUM> passes through the specific location. Therefore, based on the output of a fluorescence signal based on the passage of the sample <NUM>, for example, by controlling the driver for driving the liquid feeding system <NUM>, it is possible to perform the feeding of the sample <NUM> in a reciprocating manner accompanied by proper positioning of the sample <NUM>. Further, a plurality of optical heads of the fluorescence detector <NUM> can be arranged along the channel <NUM>. For example, by arranging an optical head of the fluorescence detector <NUM> immediately below each reaction region, the presence or absence of the sample <NUM> in each reaction region can be detected, thus allowing for more reliable positioning of the sample <NUM>.

The reaction processing method in which the reaction processor <NUM> according to the second embodiment is used is advantageous in that, unlike the reaction processing method in which the reaction processor <NUM> is used according to the first embodiment, fluorescence from the sample <NUM> can be continuously detected even during the reaction process by means of the thermal cycle and the progress of DNA amplification can be managed in real time as described above.

As described above, in the reaction processor <NUM> according to the second embodiment, the pressure inside the channel <NUM> of the reaction processing vessel <NUM> is always maintained to be higher than the air pressure in the surrounding environment of the reaction processor <NUM>, preferably <NUM> atm or higher, during the reaction process. In other words, during the reaction process, the sample <NUM> is constantly pressurized to have a pressure higher than the air pressure in the surrounding environment of the reaction processor <NUM>, preferably <NUM> atm or higher. Therefore, stable PCR can be performed while preventing the boiling of a sample and the generation of air bubbles even in a place where the air pressure is low such as a high altitude place or the inside of an airplane.

<FIG> is a schematic diagram for explaining a reaction processor <NUM> according to the third embodiment of the present invention. In the reaction processor <NUM> according to the third embodiment, since a reaction processing vessel that is the same as the reaction processing vessel <NUM> (see <FIG>) described in the second embodiment is used, like numerals represent like constituting elements, and duplicative explanations will be omitted. Further, since a temperature control system and a fluorescence detector that are the same as the temperature control system <NUM> and the fluorescence detector <NUM> explained in the second embodiment are also used in the reaction processor <NUM>, like numerals represent like constituting elements, and duplicative explanations will be omitted. In the reaction processor <NUM> according to the third embodiment of the present invention, the configuration of the liquid feeding system and the reaction processing method based thereon are different from those according to the second embodiment.

A liquid feeding system <NUM> of the reaction processor <NUM> according to the third embodiment of the present invention is provided with a liquid feeding chamber <NUM>, a pressurizing chamber <NUM>, a liquid feeding chamber pump <NUM>, a liquid feeding chamber pump driver <NUM> for controlling the liquid feeding chamber pump <NUM>, a pressurizing chamber pump <NUM>, a pressurizing chamber pump driver <NUM> for controlling the pressurizing chamber pump <NUM>, a first direction switching valve <NUM>, a second direction switching valve <NUM>, a first tube <NUM>, and a second tube <NUM>. Also, the reaction processor <NUM> may be provided with a driver (not shown) for controlling the first direction switching valve <NUM> and the second direction switching valve <NUM>.

<FIG> is a schematic diagram for explaining the configuration of a direction switching valve. The direction switching valve <NUM> shown in <FIG> can be used as the first direction switching valve <NUM> and the second direction switching valve <NUM> in the reaction processor <NUM> shown in <FIG>. As shown in <FIG>, the direction switching valve <NUM> is provided with a first supply port <NUM>, a second supply port <NUM>, and a discharge port <NUM>. The direction switchingvalve <NUM> is capable of switching communication between the first supply port <NUM> and the discharge port <NUM> and communication between the second supply port <NUM> and the discharge port <NUM>. A means for the switching of communication may be of a direct acting electromagnetic type or pilot electromagnetic type in which an internal valve is switched by a separate air pressure. Also, a so-called universal type valve may be used, which allows the air to flow bidirectionally in a path between the first supply port <NUM> and the discharge port <NUM> or a path between the second supply port <NUM> and the discharge port <NUM>. Alternatively, a direction switching valve provided with four or more ports can be also used. Further, the direction switching valve is not limited to these and may be something like a three-way valve. Further, the valve maybe provided with a structure that controls the rotation of the three-way cock with a stepping motor or the like.

Referring back to <FIG>, a first end portion 246a of the first tube <NUM> is connected to the first communication port <NUM> of the reaction processing vessel <NUM>. A packing material or a seal for securing airtightness is preferably arranged at the connection between the first communication port <NUM> and the first end portion 246a of the first tube <NUM>. The second end portion 246b of the first tube <NUM> is connected to the discharge port of the first direction switching valve <NUM>. Further, the first supply port of the first direction switching valve <NUM> is connected to the liquid feeding chamber <NUM> by a hollow tube <NUM>. Further, the second supply port of the first direction switching valve <NUM> is connected to the pressurizing chamber <NUM> by a hollow tube <NUM>.

In the same way, a first end portion 247a of the second tube <NUM> is connected to the second communication port <NUM> of the reaction processing vessel <NUM>. A packing material or a seal for securing airtightness is preferably arranged at the connection between the second communication port <NUM> and the first end portion 247a of the second tube <NUM>. The second end portion 247b of the second tube <NUM> is connected to the discharge port of the second direction switching valve <NUM>. Further, the first supply port of the second direction switching valve <NUM> is connected to the liquid feeding chamber <NUM> by a hollow tube <NUM>. Further, the second supply port of the second direction switching valve <NUM> is connected to the pressurizing chamber <NUM> by a hollow tube <NUM>.

The liquid feeding chamber <NUM> forms a space having a certain volume therein. The liquid feeding chamber pump <NUM> is connected to the liquid feeding chamber <NUM>. The liquid feeding chamber pump driver <NUM> controls the liquid feeding chamber pump <NUM> such that the space inside the liquid feeding chamber <NUM> has a predetermined pressure in accordance with an instruction from a CPU <NUM>.

In the same way, the pressurizing chamber <NUM> forms a space having a certain volume therein. The pressurizing chamber pump <NUM> is connected to the pressurizing chamber <NUM>. The pressurizing chamber pump driver <NUM> controls the pressurizing chamber pump <NUM> such that the space inside the pressurizing chamber <NUM> has a predetermined pressure in accordance with an instruction from the CPU <NUM>.

As the liquid feeding chamber pump <NUM> and as the pressurizing chamber pump <NUM>, a compact DC diaphragm pump (DSA-<NUM>-12BL model) manufactured by Denso Sangyo Co. , or the like can be used, and a means of pressurization by a rubber ball, a syringe, or the like can be also used as a simple means.

In the third embodiment, the pressure inside the liquid feeding chamber <NUM> and the pressure inside the pressurizing chamber <NUM> are set to be higher than the air pressure in the surrounding environment of the reaction processor <NUM> during the reaction process and more preferably maintained at <NUM> atm (<NUM> hPa) or higher. The pressure inside the liquid feeding chamber <NUM> is maintained to be higher than the pressure inside the pressurizing chamber <NUM> during the reaction process.

The liquid feeding chamber <NUM> and the pressurizing chamber <NUM> are provided with atmospheric pressure releasing valves <NUM> and <NUM>, respectively. By the atmospheric pressure releasing valves <NUM> and <NUM>, the pressure condition inside the chamber can be reset when the reaction processor <NUM> is repeatedly used such that sudden movement and squirting of the sample <NUM> at the time of installing or removing the reaction processing vessel <NUM> can be prevented.

A description will be given of a reaction processing method in which the reaction processor <NUM> configured as described above is used. In the initial state of the processor, it is assumed that the second end portion 246b of the first tube <NUM> is connected to the discharge port of the first direction switching valve <NUM> and that the first end portion 246a of the first tube <NUM> is open. Also, it is assumed that the second end portion 247b of the second tube <NUM> is connected to the discharge port of the second direction switching valve <NUM> and that the first end portion 247a of the second tube <NUM> is open.

Next, the atmospheric pressure releasing valves <NUM> and <NUM> are opened such that the respective pressures in the liquid feeding chamber <NUM>, the pressurizing chamber <NUM>, the first direction switching valve <NUM>, the second direction switching valve <NUM>, the first tube <NUM>, and the second tube <NUM> become equal to the atmospheric pressure. Subsequently, the first end portion 246a of the first tube <NUM> extending from the first direction switching valve <NUM> is connected to the first communication port <NUM> of the reaction processing vessel <NUM>, and the first end portion 247a of the second tube <NUM> extending from the second direction switching valve <NUM> is connected to the second communication port <NUM> of the reaction processing vessel <NUM>. Neither the liquid feeding chamber pump <NUM> nor the pressurizing chamber pump <NUM> is operated at this point. Subsequently, the atmospheric pressure releasing valves <NUM> and <NUM> are closed.

Next, after switching to the respective paths where the second supplyports communicating with the pressurizing chamber <NUM> communicate with the respective discharge ports by operating the first direction switching valve <NUM> and the second direction switching valve <NUM>, the pressurizing chamber pump <NUM> is operated. The pressure in the pressurizing chamber <NUM> is increased to be higher than the air pressure in the surrounding environment of the reaction processor <NUM> and more preferably to be <NUM> atm (<NUM> hPa) or higher. The pressurizing chamber <NUM> communicates with the first communication port <NUM> and the second communication port <NUM> of the reaction processing vessel <NUM> via the respective second supply ports and the respective discharge ports of the first direction switching valve <NUM> and the second direction switching valve <NUM>. Therefore, since the pressures on respective sides (the first communication port <NUM> side and the second communication port <NUM> side) of the sample <NUM> also become equal due to the increasing of the pressure in the pressurizing chamber <NUM>, the pressure balance in the channel is not affected, and the sample <NUM> thus does not move.

Subsequently, the temperature control system <NUM> is operated so as to start the temperature control of each temperature region in the reaction processing vessel <NUM>. The temperature control may be put on hold for a predetermined amount of time until the temperature in each temperature region is stabilized.

Subsequently, the liquid feeding chamber pump <NUM> is operated to raise the pressure inside the liquid feeding chamber <NUM>. Since the liquid feeding chamber <NUM> does not communicate with the spaces on respective sides (the first communication port <NUM> side and the second communication port <NUM> side) of the sample <NUM> in the channel of the reaction processing vessel <NUM> at this point, the pressure in the channel is not affected by the increasing of the pressure in the liquid feeding chamber <NUM>, and the sample <NUM> thus does not move. However, the pressure inside the liquid feeding chamber <NUM> is higher than the pressure inside the pressurizing chamber <NUM> as described above. This pressure difference serves as a propulsive force for moving the sample <NUM>.

It is assumed that the initial position of the sample <NUM> is located, for example, in the high temperature region <NUM> shown in <FIG>. When the sample <NUM> is in the high temperature region <NUM> for a certain period of time, denaturation of the DNA occurs. First, the first direction switching valve <NUM> is operated to switch to a path where the first supply port and the discharge port communicate with each other. Thereby, the pressure in the space on the first communication port <NUM> side of the sample <NUM> becomes equal to the pressure inside the liquid feeding chamber <NUM>, and the pressure in the space on the first communication port <NUM> side becomes higher than the pressure on the second communication port <NUM> side. Thus, the sample <NUM> can move from the high temperature region <NUM> to the low temperature region <NUM> via the medium temperature region <NUM> while being pushed inside the channel <NUM> toward the second communication port <NUM>. When the sample <NUM> reaches the low temperature region <NUM>, the first direction switching valve <NUM> is operated to switch to a path where the second supply port and the discharge port communicate with each other. Thereby, the pressure in the space on the first communication port <NUM> side of the sample <NUM> becomes the same as the pressure inside the pressurizing chamber <NUM>, and the pressure in the space on the first communication port <NUM> side becomes equal to the pressure on the second communication port <NUM> side. Thus, the sample <NUM> stops moving. Placing the sample <NUM> in the low temperature region <NUM> for a certain period of time causes annealing of the DNA.

Subsequently, the second direction switching valve <NUM> is operated to switch to a path where the first supply port and the discharge port communicate with each other. Thereby, the pressure in the space on the second communication port <NUM> side of the sample <NUM> becomes the same as the pressure inside the liquid feeding chamber <NUM>, and the pressure in the space on the second communication port <NUM> side becomes higher than the pressure on the first communication port <NUM> side. Thus, the sample <NUM> can move from the low temperature region <NUM> to the medium temperature region <NUM> while being pushed inside the channel toward the first communication port <NUM>. When the sample <NUM> reaches the medium temperature region <NUM>, the second direction switching valve <NUM> is operated to switch to a path where the second supply port and the discharge port communicate with each other. Thereby, the pressure in the space on the second communication port <NUM> side of the sample <NUM> becomes the same as the pressure inside the pressurizing chamber <NUM>, and the pressure in the space on the second communication port <NUM> side becomes equal to the pressure on the first communication port <NUM> side. Thus, the sample <NUM> stops moving. Placing the sample in the medium temperature region <NUM> for a certain period of time causes elongation of the DNA.

Further, the second direction switching valve <NUM> is operated to switch to a path where the first supply port and the discharge port communicate with each other. Thereby, the sample <NUM> can move from the medium temperature region <NUM> to the high temperature region <NUM> while being pushed inside the channel <NUM> toward the first communication port <NUM>. When the sample <NUM> reaches the high temperature region <NUM>, the second direction switching valve <NUM> is operated to switch to a path where the second supply port and the discharge port communicate with each other. Thereby, the pressure in the space on the second communication port <NUM> side of the sample <NUM> becomes the same as the pressure inside the pressurizing chamber <NUM>, and the pressure in the space on the second communication port <NUM> side becomes equal to the pressure on the first communication port <NUM> side. Thus, the sample <NUM> stops moving. Placing the sample in the high temperature region <NUM> for a certain period of time causes denaturation of the DNA.

By controlling the operation of the first direction switching valve <NUM> and the second direction switching valve <NUM> of the liquid feeding system <NUM> so as to repeat the movement of the sample <NUM> described above, the sample <NUM> is allowed to reciprocate inside the channel <NUM>. More specifically, the sample <NUM> cyclically passes through the respective regions of the temperatures: a high temperature (denaturation); a low temperature (annealing); a medium temperature (elongation) ; a high temperature (denaturation); a low temperature (annealing); a medium temperature (elongation); and so on. Further, in the case of a reaction processor where temperature regions of two levels are set, the sample <NUM> cyclically passes through the respective regions of the temperatures: a high temperature (denaturation); a medium-low temperature (annealing and elongation); a high temperature (denaturation); a medium-low temperature (annealing and elongation); and so on. This allows a predetermined number of thermal cycles to be applied to the sample <NUM> and allows PCR to occur such that predetermined DNA can be selectively amplified.

As described above, in the reaction processor <NUM> according to the third embodiment, the pressure inside the channel <NUM> of the reaction processing vessel <NUM> is always maintained to be higher than the air pressure in the surrounding environment of the reaction processor <NUM>, more preferably <NUM> atm or higher, during the reaction process. In other words, the sample <NUM> is constantly pressurized to be higher than the air pressure in the surrounding environment maintained in the pressurizing chamber <NUM>, more preferably <NUM> atm (<NUM> hPa) or higher, or pressurized by the pressure in the liquid feeding chamber <NUM>, which is higher than the pressure in the pressurizing chamber <NUM>. Therefore, PCR can be performed while preventing the boiling of a sample and the generation of air bubbles even in a place where the air pressure is low such as a high altitude place or the inside of an airplane.

The reaction processors according to the present invention have been explained above. In the case of a reaction processor unrelated to the reaction processors according to the present invention, that is, in the case of a reaction processor according to an embodiment where a sample in a channel is not pressurized, the sample may easily boil and/or foam when PCR is performed under a low air pressure environment such as a high altitude place or the inside of an airplane. Foamed bubbles are often generated in the middle of the sample, and a plurality of bubbles are often generated. In that case, a phenomenon occurs in which the pressure in the bubbles generated between pieces of the sample and the pressure for liquid feeding and the like start to be balanced such that the liquid feeding cannot be performed smoothly, e.g., a part of the sample stops in the channel.

According to the reaction processors according to the present invention described above, the probability of foaming can be drastically reduced in the first place such that the above problem does not arise and, even under any air pressure environment, the feeding of a sample can be performed in an almost perfect manner, and as a result, amplified samples such as DNA can be obtained through a stable reaction process.

Described above is an explanation of the present invention based on the embodiments. These embodiments are intended to be illustrative only, the scope of the invention being defined by the appended claims.

Claim 1:
A reaction processor (<NUM>) for performing a reaction process of a sample by applying a thermal cycle to the sample while the sample moves inside a channel (<NUM>) of a reaction processing vessel (<NUM>), the reaction processor comprising:
a liquid feeding system (<NUM>) for controlling the pressure inside the channel (<NUM>) of the reaction processing vessel (<NUM>) in order to move and stop the sample inside the channel (<NUM>),
wherein the liquid feeding system (<NUM>) includes:
a pressurizing chamber (<NUM>);
a first liquid feeding pump (<NUM>) that is arranged inside the pressurizing chamber (<NUM>); and
a second liquid feeding pump (<NUM>) that is arranged inside the pressurizing chamber (<NUM>),
wherein the output of the first liquid feeding pump (<NUM>) communicates with a first communication port (<NUM>) that is provided at one end of the channel (<NUM>) of the reaction processing vessel (<NUM>), and
wherein the output of the second liquid feeding pump (<NUM>) communicates with a second communicationport (<NUM>) that is provided at the other end of the channel (<NUM>) of the reaction processing vessel (<NUM>),
wherein the liquid feeding system (<NUM>) further comprises a control unit (<NUM>) for controlling the first liquid feeding pump (<NUM>) and the second liquid feeding pump (<NUM>) in order to move the sample inside the channel (<NUM>), and
wherein the liquid feeding system (<NUM>) is arranged to maintain the pressure inside the channel (<NUM>) to be higher than the air pressure in the surrounding environment of the reaction processor (<NUM>) during the reaction process of the sample by maintaining the internal pressure inside the pressurizing chamber (<NUM>) to be higher than the air pressure in the surrounding environment of the reaction processor (<NUM>).