JP5384903B2 - Temperature control reaction processing apparatus and temperature control reaction processing method - Google Patents

Description

  The present invention relates to a temperature controlled reaction processing apparatus and a temperature controlled reaction processing method.

  In recent years, as a DNA amplification method for rapidly and easily amplifying a specific DNA fragment, a polymerase chain reaction (PCR) method and a temperature control process such as a constant temperature enzyme reaction have been carried out in all biological fields. ing. In particular, the PCR method is a method in which two primers complementary to a template DNA are designed, and a region sandwiched between the primers is replicated in vitro (in vitro). In this method, PCR products are obtained by amplifying DNA exponentially by repeating temperature cycles of incubating a reaction solution containing template DNA, primers, nucleotides, and heat-resistant DNA polymerase at various temperatures.

In one cycle, for a container containing template DNA, primer, DNA polymerase, nucleotide and reaction buffer solution, double-stranded DNA is denatured to single-stranded, and the primer anneals to single-stranded DNA. It consists of incubating under the respective temperature conditions (94 ° C., 50 ° C. to 60 ° C., and 74 ° C., respectively) for synthesizing a DNA strand complementary to a single strand, so that one molecule of DNA fragment is made into two molecules. In the next cycle, the DNA fragment synthesized in the previous cycle also serves as a template, so the DNA fragment synthesized after n cycles becomes 2 ⁿ molecules.

  Conventionally, the temperature is controlled by placing a container made of glass or the like containing the template DNA, primer, DNA polymerase, nucleotide, reaction buffer solution, etc. in a storage room of a block-shaped thermostat made of a material such as aluminum. The metal block-shaped container is heated or cooled and waits until the liquid temperature has a uniform temperature distribution, thereby heating or cooling the next temperature (Patent Document). 1, 2, 3).

  At that time, sealing the container for performing the temperature control reaction process with a lid prevents intrusion of foreign matter from the outside, prevents liquid leakage from the inside, and heats the reaction liquid in the storage section. It is particularly necessary to eliminate as much as possible the influence of outside air and outside air temperature until it is cooled and has a uniform liquid temperature distribution.

  However, conventionally, since the user manually performs sealing of the container with a lid, it takes time and hinders the consistent automation of the process. Further, since the user performs it manually, there is a risk of contamination due to contact with the reaction solution in the container when the lid is sealed with the container. Furthermore, even if the lid is removed from the container after the reaction is completed, the lid may be in close contact with the opening of the container due to moisture, making it difficult to open the lid, and there is a possibility that rapid processing cannot be performed. It was. In addition, when the lid is opened, the liquid adhering to the inside of the lid may drip or scatter, which may cause contamination (Patent Document 4).

Japanese Patent No. 2622327 Special Table 2000-511435 US Pat. No. 5,958,349 JP 2002-10777 A

  Therefore, the present invention has been made to solve the above problems, and a first object of the present invention is to reliably and quickly close and open the opening of the reaction vessel with an upper lid to analyze the reaction product. To provide a temperature-controlled reaction processing apparatus and a temperature-controlled reaction processing method that have high processing efficiency that can be used quickly, and that can be manufactured and used at low cost without increasing the scale of the apparatus. It is.

  The second purpose is to provide an upper lid on the reaction vessel whose temperature is controlled, and to allow the upper lid to close and open the opening of the reaction vessel, and to perform other actions on the upper lid without manual operation. Temperature-controlled reaction processing apparatus and temperature-controlled reaction processing capable of reliably preventing contamination due to entry of foreign matter from the outside into the reaction vessel, liquid leakage from the reaction vessel, etc., and performing highly reliable processing It is intended to provide a method.

  The third purpose is to provide an upper lid in a reaction vessel whose temperature is controlled and press the upper lid to reliably prevent liquid leakage and gas leakage, or to allow the upper lid to be shaken or moved when the upper lid is closed. Thus, it is an object of the present invention to provide a temperature-controlled reaction processing apparatus and a temperature-controlled reaction processing method that reduce the user's trouble by opening the upper lid reliably and easily from the reaction container.

  A first invention has a suction / discharge mechanism for sucking and discharging gas, and a nozzle head having one or more nozzles to which a dispensing tip capable of sucking and discharging liquid can be detachably mounted by the suction / discharge mechanism One or more reaction containers having an opening into which the tip of the dispensing tip can be inserted, a moving mechanism that allows relative movement between the nozzle and the reaction container, and one or more reaction containers A temperature controller capable of controlling the temperature inside the reaction vessel, an upper lid provided to be openable and closable with respect to the opening of one or more of the reaction vessels whose temperature is controlled by the temperature controller, and the suction discharge A temperature control reaction processing apparatus having a mechanism for closing the upper lid that enables the upper lid to be pressed, shaken or moved when the opening is closed by the upper lid by the mechanism or the moving mechanism.

  In order to “removably attach”, for example, it is preferable to provide a desorption mechanism for desorbing the dispensing tip from the nozzle on a stage provided with a nozzle head or a reaction vessel. In order to attach the dispensing tip to the nozzle, for example, it is accommodated in the tip accommodating portion provided on the stage with the nozzle mounting opening of the dispensing tip facing upward. The nozzle can be mounted by inserting the lower end of the nozzle into the opening for mounting the nozzle by moving the nozzle and the tip accommodating portion in a direction of approaching by the vertical movement mechanism of the moving mechanism. The nozzle head is preferably provided with a magnetic means capable of exerting and removing a magnetic force in the mounted dispensing tip in order to extract a substance such as DNA from a specimen.

  Here, as the “desorption mechanism”, for example, a plate having a hole having a diameter larger than the outer diameter of the nozzle but smaller than the thickest part of the dispensing tip is provided in the nozzle head or the stage, There is a mechanism for removing the mounted dispensing tip by lowering the plate along the axial direction of the nozzle penetrating each hole. When the plate is provided on the nozzle head, the attachment / detachment is performed by interlocking the plate with the drive mechanism of the piston that slides in the cylinder as the suction / discharge mechanism, and the plate is provided on the stage. In this case, the nozzle is moved in the vertical direction relative to the plate by the vertical movement mechanism.

  "Because it is relatively movable between the nozzle and the reaction vessel", when the reaction vessel or stage is fixed and the nozzle is moved, and when the nozzle is fixed and the stage or reaction vessel is moved, And a combination of both.

  In addition to the reaction container, the stage is preferably provided with a liquid storage unit for storing a liquid such as a specimen and a reagent, and a chip storage unit for storing a chip such as the dispensing tip. In addition, the container includes a microplate in which wells as a plurality of liquid storage portions are arranged in a matrix or a column (row), and a cartridge-like container in which wells as a plurality of liquid storage portions are arranged in a row. . The nozzle or the dispensing tip attached to the nozzle reaches these containers by the moving mechanism, and can suck and discharge liquid, or attach and detach the tip.

  Examples of materials such as a container including the reaction container and an upper lid include resins such as polyethylene, polypropylene, polystyrene, and acrylic, glass, metal, and metal compounds. The size of the container is, for example, a size that can accommodate a liquid of several μl to several hundreds of μl and can be inserted into the tip of the dispensing tip. For example, in the case of a cylindrical shape, for example, the diameter of the size of one container is several millimeters to several tens of millimeters, and the depth is several millimeters to several tens of millimeters.

  The “dispensing tip” includes, for example, a large-diameter portion, a small-diameter portion, and a transition portion that communicates the large-diameter portion and the small-diameter portion. It has a mounting opening to be inserted and mounted on the nozzle, and the narrow-diameter portion has a front end opening through which liquid can flow in and out by suction and discharge of gas by the suction and discharge mechanism. Dispensing tips and nozzles are made of, for example, organic materials such as resins such as polypropylene, polystyrene, polyester, polystyrene, and acrylic, metals such as glass and ceramics, stainless steel, and the like, inorganic compounds such as metal compounds, and semiconductors. In addition, it can replace with a dispensing chip | tip, and it can also process by mounting | wearing the said nozzle with the analysis chip | tips, such as a particulate carrier enclosure chip | tip mentioned later.

  The “temperature controller” has a temperature source capable of raising or lowering the temperature in the reaction container containing the liquid to be temperature controlled based on an external signal or the like. For example, there are Peltier elements, heaters, cooling devices, and the like. In order to perform a process such as PCR, a thermal cycler using a Peltier element is preferable as the temperature controller.

  “Temperature control” means that a target liquid or container is maintained at one or two or more set predetermined temperatures for a set time in accordance with a set order. It is. The temperature controller is instructed by sending a corresponding signal based on a program.

  The “predetermined temperature” is a target temperature that an object such as a target liquid should reach. For example, when a nucleic acid such as DNA or an oligonucleotide contained in the liquid is amplified by a PCR method, The predetermined temperature to be set is, for example, a temperature cycle performed by the PCR method, that is, each temperature required for DNA denaturation, annealing or hybridization, and extension, approximately 94 ° C., between 50 ° C. and 60 ° C. For example, about 50 ° C. and about 72 ° C. Furthermore, for example, in the case of a transition from a predetermined temperature at a high temperature to a predetermined temperature at a low temperature, the predetermined temperature can be cooled by a temperature regulator at a transition promoting temperature lower than the predetermined temperature. Or, at the time of transition from a predetermined temperature of low temperature to a predetermined temperature of high temperature, heating is performed at a temperature for promoting transition higher than these predetermined temperatures, thereby shortening the transition time and reducing one cycle time. Includes transition-promoting temperature to keep within predetermined cycle time. “Predetermined time” is the time required to maintain each temperature, and depends on the reagent and volume used in the PCR method, the shape, material, size, thickness, etc. of the nozzle. For example, the processing time of the entire PCR method is about several minutes to several tens of minutes, for example, from several seconds to several tens of seconds. The transition time is also included in the predetermined time.

  The “suction / discharge mechanism” includes, for example, a cylinder, a piston sliding in the cylinder, a nut portion connected to the piston, a ball screw into which the nut portion is screwed, and the ball screw in both forward and reverse directions. It has a motor that rotates and a pump mechanism.

  As the “movement mechanism”, for example, there is a mechanism that enables movement between the reaction vessel, that is, a stage in which the reaction vessel is provided, and a nozzle relatively in an axial direction and a horizontal plane of the nozzle. As the movement in the horizontal plane, for example, the stage or nozzle moves along the X axis and the Y axis, the XY axis movement mechanism that moves, or the Y (X) axis movement mechanism that moves only along the Y axis or the X axis. As the movement of the nozzle in the axial direction, there is a vertical movement mechanism provided in the nozzle head that moves the nozzle in the axial direction (Z-axis direction). The operating tool is driven by this moving mechanism so as to interlock with the nozzle.

  Since the upper lid can be pressed, shaken or moved when the opening is closed, the upper lid is not fixed to the opening in a stationary state, but a certain amount of relative movement is present between the opening and the upper lid. It is possible to move. The pressing, shaking, or moving may be performed by, for example, pressing, shaking, or moving from above the upper lid, from the horizontal direction of the upper lid, or from a direction other than the upper direction and the horizontal direction.

  According to a second aspect of the present invention, the suction / discharge mechanism includes a cylinder communicating with the nozzle, a piston that slides within the cylinder, and a piston drive mechanism that drives the piston. The temperature control reaction processing apparatus is provided in the nozzle head and has one or more operating tools interlocking with the piston. Here, the “actuator” is an instrument for applying a force to the upper lid, and has a member provided separately from the upper lid, for example, a rod shape, a cylindrical shape, a cone shape, etc. The end of the member is a portion that can come into contact with the upper lid.

  A third aspect of the invention is a temperature control reaction processing apparatus in which the upper lid closing mechanism is provided in the nozzle head and has one or more operating tools interlocking with the nozzle driven by the moving mechanism.

  In the case of the second invention and the third invention, it is preferable that the axial direction of an operating tool such as a rod and the axial direction of the nozzle are normally aligned with the vertical direction.

  In addition, when it has two or more actuators, about an up-down direction movement, a part of actuator may be driven by the said movement mechanism, and another actuator may be driven by the said piston drive mechanism. good.

  Since the operating tool is driven by a moving mechanism or a piston driving mechanism, it is not necessary to provide a new mechanism for driving the operating tool. The actuator can be driven with similar strength.

  In addition, it is preferable that the horizontal movement other than the vertical movement is configured such that the entire nozzle head is moved by a moving mechanism provided on a stage or casing outside the nozzle head. In this case, since all the operating tools are provided in the nozzle head, horizontal movement other than vertical movement moves for each nozzle head.

  4th invention is a temperature control reaction processing apparatus in which the action mechanism at the time of closing the upper lid has one or more nozzles driven by the moving mechanism.

  In the case of the second, third, or fourth invention, it is preferable that the detaching mechanism for detaching the dispensing tip attached to the nozzle is provided in the nozzle head, and the dispensing tip is previously detached by the detaching mechanism. . As a result, it is possible to prevent the dispensing tip from dropping off or the remaining liquid from the dispensing tip from splashing when the actuator is driven, as well as the components linked to drive the actuator and nozzle. Since it is not necessary to consider the space in which the tip can move, the work space can be used efficiently to increase the work efficiency.

  5th invention is a temperature control reaction processing apparatus which has the hook which can be engaged with the said operation tool or the said nozzle by the said moving mechanism or the said suction discharge mechanism on the upper surface of the said upper cover.

  Here, as the “hook”, a plate in which a lower end portion of the working tool and a lower end portion of the nozzle can be fitted and a U-shaped cutout portion and a slit are provided. . In order to engage the operating tool or nozzle with the hook, a convex portion or a concave portion may be provided at the lower end of the operating tool or nozzle. In this case, the “hook” is, for example, a substantially U-shaped notch or slit formed by notching a plate provided apart from the upper surface. The “convex portion” is, for example, a flange provided along the outer periphery of the lower end portion of the operating tool or nozzle, and the “recessed portion” is, for example, along the outer periphery of the lower end portion of the operating tool or nozzle. The groove is provided, and the flange is located below the plate, and the notch and the slit are formed in such a size that the flange cannot pass, or the notch and the slit are fitted into the groove. Provide as possible.

  In order to engage the operating tool or the nozzle with the hook, for example, the operating tool or the nozzle is moved in a horizontal plane having a predetermined height by using the moving mechanism, so that the notch included in the hook is provided. The actuator or the nozzle and the hook are engaged by inserting from the side where the part or the slit is opened. In addition, when a convex part and a recessed part are provided, a connection with the said hook can be performed reliably and firmly. In particular, the upper lid can be moved upward.

  The sixth invention further includes a shaft support portion attached to a stage provided with the reaction vessel, and an upper lid support member that is pivotally supported by the shaft support portion and elastically supports the upper lid. A pressed surface to be pressed by the action mechanism at the time of closing the upper lid, and a covering surface formed so as to face the pressed surface and covering the opening of the reaction vessel, and the upper lid support member covers the upper lid with the coated surface It is the temperature control reaction processing apparatus supported in the state which elastically biased along the direction which goes to the said to-be-pressed surface. In the coated state, the coated surface preferably floats from the opening.

  Here, the “shaft support portion” is a member that rotatably supports the upper lid, and includes, for example, a fixing block fixed on the stage, an open / close hinge provided on the fixing block, and a rotating shaft, The upper lid is pivotally supported via the upper lid support member. In order for the pressed surface to be pressed from the outside, it is necessary to expose at least the pressed surface outward (upward in the following embodiment) when the reaction container of the upper lid is closed.

  When the upper lid is formed in a hollow box shape or a cylindrical shape provided with the pressed surface and the covering surface, the hollow portion provides a heat insulating effect to the reaction vessel whose temperature is controlled. Thereby, a uniform temperature distribution can be given to the liquid stored in the reaction vessel. Furthermore, a heat source such as a heater can be provided in the hollow portion to prevent dew condensation occurring on the cover surface of the upper lid. Thus, when the upper lid is opened, it is possible to prevent adhesion between the covering surface of the upper lid and the reaction vessel due to condensation.

  In addition, by providing an opening / closing drive motor, the upper lid supported by the shaft support portion can be rotationally driven to open / close with respect to the opening portion. In this case, a series of operations including the opening / closing operation of the upper lid can be performed without touching, and contamination can be reliably prevented.

  7th invention performs the suction / discharge of the gas provided in the movement mechanism and nozzle head which can move relatively between the 1 or 2 or more nozzle provided in the nozzle head, and 1 or 2 or more reaction containers. With the suction and discharge mechanism, the temperature of the liquid sucked into the dispensing tip detachably attached to one or two or more of the nozzles can be controlled, and the upper lid that can be opened and closed with respect to the opening is opened. An accommodating step of discharging and storing in one or two or more of the reaction containers, and an opening of one or more of the reaction containers are closed by the upper lid, and the upper lid is moved upward by the suction / discharge mechanism or the moving mechanism. And the upper lid closed with respect to the opening of the reaction vessel is shaken or moved by the closing step of controlling the temperature inside the reaction vessel by pressing and the suction / discharge mechanism or the moving mechanism. A releasing step of releasing the opening of the reaction vessel by a temperature control reaction treatment method with.

  According to an eighth aspect of the present invention, the closing step is performed by using one or more operating tools driven by the suction / discharge mechanism or by the moving mechanism, and is provided in the nozzle or the nozzle head. Using one or more operating tools interlocking with the nozzle, the upper lid that closed the opening is pressed from above, and the opening step closes the opening using the nozzle or the operating tool. This is a temperature control reaction processing method in which the upper lid is shaken or moved.

  Here, “to drive one or more actuators by the suction / discharge mechanism” means, for example, 1 or 2 that is linked to the piston by a piston drive mechanism that drives a piston that slides in a cylinder communicating with the nozzle. In some cases, the above-described operating tool is driven.

  The ninth invention further includes a desorption step of desorbing the dispensing tip from the nozzle, and the desorption step is performed in the accommodating step or after the accommodating step and before the closing step. This is a temperature control reaction processing method.

  A tenth aspect of the invention is a temperature control reaction processing method in which the accommodation step is performed by attaching one dispensing tip to only one nozzle among the plurality of nozzles arranged in the nozzle head. . Here, the “one nozzle” is preferably a nozzle provided at the end of the nozzle row arranged in the nozzle head. In this case, even if chips other than one chip to be mounted are also arranged, only one chip can be reliably mounted on one nozzle. From the second time onward, it is also possible to attach one chip to other nozzles by sequentially removing the used chips from the nozzle and the chip housing portion.

  According to the first invention or the seventh invention, an existing mechanism provided in a dispensing device such as a suction / discharge mechanism or a moving mechanism is provided while providing an openable / closable upper lid on one or more reaction containers capable of controlling temperature. The upper lid with the opening of the reaction vessel closed can be pressed, shaken, and the like.

  Therefore, by pressing, shaking, or moving the upper lid in a state where the opening is closed, the opening is reliably closed with the upper lid, or the sealed state between the opening and the upper lid is quickly closed. And it can be easily released and opened.

  Therefore, high processing efficiency and reliability can be obtained. Further, it can be manufactured and used at low cost without increasing the scale of the apparatus.

  By performing an action such as pressing on the upper lid in a state in which the opening is closed without performing the manual operation, foreign substances can be reliably prevented from entering the reaction vessel. Furthermore, by removing the moisture due to condensation attached to the surface of the upper lid by shaking or moving the upper lid in a state in which the opening is closed while applying a force parallel to the surface of the upper lid in contact with the opening. Can do. Therefore, it is possible to reliably prevent contamination due to entry of foreign matter into the reaction vessel or leakage of liquid from the reaction vessel when the upper lid is opened, so that highly reliable processing can be performed. Moreover, it is easy to handle by reducing the user's trouble.

  In addition, by simultaneously closing a plurality of reaction vessels with one upper lid, temperature control can be performed efficiently for a plurality of types of target liquids in parallel.

  According to 2nd invention or 3rd invention, the said operation tool is provided so that it may interlock | cooperate with the said piston or a nozzle. Accordingly, the operating tool can easily and reliably act on the upper lid using an existing mechanism. Therefore, it can be manufactured at low cost without increasing the scale of the apparatus.

  In addition, when an operating tool that is a dedicated instrument that acts on the upper lid is provided, contamination due to the action with the upper lid can be more reliably prevented and reliability is higher than when a nozzle is used. In addition, since the operating tool can use either the moving mechanism or the piston drive mechanism for each operating tool, it is possible to cause the upper lid to act stably and strongly by using both mechanisms simultaneously.

  According to the fourth or eighth invention, since the nozzle and the moving mechanism can be used to act on the upper lid when the opening of the reaction vessel is closed, the scale of the apparatus can be obtained using only the existing mechanism. It is possible to manufacture at a lower cost without enlarging.

  According to a fifth aspect of the present invention, a hook that can be engaged with a lower end portion of the operating tool or the nozzle using a moving mechanism or a suction / discharge mechanism is provided, and the operating tool or the nozzle is securely and easily attached to the upper lid. Can be linked or combined. Therefore, it is possible to connect the nozzle or the operation tool to the upper lid and to reliably apply a complicated action to the upper lid.

  Further, since the user can perform the operation without touching it directly, contamination can be prevented and highly reliable processing can be performed.

  According to the sixth aspect, the upper lid support member that elastically supports the upper lid is provided on the shaft support portion provided on the stage. Therefore, the upper lid is not directly supported by the stage but is elastically supported. Therefore, with a simple structure, the upper lid is closed by pressing the upper lid while closing the opening of the reaction vessel. Or the top lid can be shaken or the top lid can be moved.

  Moreover, when a hollow part is provided in the upper lid, it is possible to prevent condensation at the opening of the reaction vessel by inserting a heating panel into the hollow part. Furthermore, since the upper lid is elastically supported by the upper lid support member that movably supports the upper lid with respect to the stage, the adhesiveness of the opening can be provided by providing a pad on the contact surface of the upper lid with the opening of the reaction vessel. Can be increased.

  According to the eighth invention, in the closing step or the opening step, the operating tool or the nozzle is used to press, shake, or move the upper lid at the closing time. Therefore, it is possible to easily act on the upper lid by using a mechanism provided in the dispensing device such as the moving mechanism or the suction / discharge mechanism. Therefore, it can be manufactured at low cost without increasing the scale of the apparatus.

  According to the ninth aspect of the invention, by further including a desorption step of detaching the dispensing tip from the nozzle, when applying an action to the upper lid, splashing of the remaining liquid of the dispensing tip, contamination of the upper lid, Note: The chip itself can be prevented from being destroyed or contaminated. The restriction of the shape and movement of the operating tool due to the dispensing tip being mounted on the nozzle can be reduced.

  According to the tenth invention, by arranging a plurality of nozzles in the nozzle head, a plurality of dispensing tips are mounted and dispensed in a previous stage extraction process for storing DNA or the like in the reaction container. The process can be efficiently performed in parallel, and in order to handle a small amount (about several μl) of liquid such as DNA in order to collectively handle the amount of liquid to be stored in a plurality of reaction vessels, By attaching only one dispensing tip, the processing can be facilitated. At that time, by making the arrangement pitch of the reaction vessels different from the arrangement pitch of the other vessels, the reaction vessels can be integrated and efficient temperature control can be performed.

It is a partial cross section front view which shows the temperature control reaction processing apparatus which concerns on the 1st Embodiment of this invention. It is a top view of the temperature control reaction processing apparatus which concerns on the 1st Embodiment of this invention. It is sectional drawing which shows the various chip | tip which concerns on the 1st Embodiment of this invention. It is a figure which shows the nozzle head which concerns on the 1st Embodiment of this invention. It is a figure which shows the state before the press of the reaction container with an upper cover which concerns on the 1st Embodiment of this invention. It is a figure which shows the state at the time of the press of the reaction container with an upper cover concerning the 1st Embodiment of this invention. It is operation | movement explanatory drawing of the temperature control reaction processing apparatus which concerns on the 1st Embodiment of this invention. It is operation | movement explanatory drawing of the temperature control reaction processing apparatus which concerns on the 1st Embodiment of this invention. It is operation | movement explanatory drawing of the temperature control reaction processing apparatus which concerns on the 1st Embodiment of this invention. It is a partial cross section front view which shows the temperature control reaction processing apparatus which concerns on the 2nd Embodiment of this invention. It is a top view of the temperature control reaction processing apparatus which concerns on the 2nd Embodiment of this invention. It is operation | movement explanatory drawing of the temperature control reaction processing apparatus which concerns on the 2nd Embodiment of this invention. It is operation | movement explanatory drawing of the temperature control reaction processing apparatus which concerns on the 2nd Embodiment of this invention. It is operation | movement explanatory drawing of the temperature control reaction processing apparatus which concerns on the 2nd Embodiment of this invention. It is operation | movement explanatory drawing which concerns on the 2nd Embodiment of this invention. It is a top view of the temperature control reaction processing apparatus which concerns on the 3rd Embodiment of this invention. It is a figure which shows the state before the press of the reaction container with an upper cover concerning the 3rd Embodiment of this invention. It is a figure which shows the state at the time of the press of the reaction container with an upper cover concerning the 3rd Embodiment of this invention. It is operation | movement explanatory drawing of the temperature control reaction processing apparatus which concerns on the 3rd Embodiment of this invention. It is operation | movement explanatory drawing of the temperature control reaction processing apparatus which concerns on the 3rd Embodiment of this invention. It is operation | movement explanatory drawing of the temperature control reaction processing apparatus which concerns on the 3rd Embodiment of this invention.

  Next, embodiments of the present invention will be described with reference to the drawings. This embodiment should not be construed as limiting the present invention unless otherwise specified. Moreover, the same thing in each Example was represented by the same code | symbol, and description was abbreviate | omitted.

FIG. 1 shows a temperature-controlled reaction processing apparatus 10 according to a first embodiment of the present invention.
The temperature control reaction processing apparatus 10 includes a suction / discharge mechanism 23 (cylinder 24 and the like) that performs gas suction / discharge, and various chips (75, 76, 80) to be described later that suck and discharge liquid by the suction / discharge mechanism 23. , Refer to FIG. 2), and a nozzle head 12 having a plurality of nozzles 22 (four in this example) arranged at a predetermined pitch (for example, 18 mm) for detachably mounting, and various containers (liquid container 40, A stage 14 provided with a chip accommodating portion 42 and the like, and a moving mechanism (20, 21) for moving the nozzle 22 relative to the stage 14. The moving mechanism has an XY-axis moving mechanism 20 mainly provided outside the nozzle head 12 and a Z-axis moving mechanism 21 as the up-and-down moving mechanism provided in the nozzle head 12.

  The nozzle head 12, the moving mechanism (20, 21), and the stage 14 are incorporated in a housing 16 whose front center is open. An operation panel 18 is provided on the upper front side of the housing 16. The operation panel 18 includes a display and an operation keyboard. The casing 16 together with the nozzle head, the moving mechanism (20, 21), the suction / discharge mechanism 23, a temperature controller (described later) provided on the stage 14, and the like. It is electrically connected to an information processing device (not shown) provided inside. The information processing apparatus includes a peripheral device including an external memory such as a CD or DVD driver in addition to a CPU and a memory. The memory includes the nozzle head 12, the suction / discharge mechanism 23, and the moving mechanism (20, 21). Therefore, a program for controlling an action mechanism 19 at the time of closing the upper lid, which will be described later, a temperature controller 100, which will be described later, and rods 88 and 90 as operating tools, is introduced. The content of the action of the action mechanism 19 at the time of closing the upper lid, that is, the manner of pressing, shaking, vibration, strength, timing, duration, etc. is, for example, the content of the instruction, the content of the temperature control, the temperature, the humidity, the upper lid and the reaction It is determined based on the size of the container or the amount of contents.

  The nozzle head 12 includes a Z-axis motor 34 mounted on a motor support 35, a ball screw 31 that is rotationally driven by the Z-axis motor 34, and a nut portion (41) that engages with the ball screw 31. And a cylinder support 25 that fixes and supports the four cylinders 24 arranged at the predetermined pitch, and moves the nozzle 22 in the Z-axis direction (vertical direction). It corresponds to 21. The lower end of the ball screw 31 is rotatably supported on the substrate 39.

  The nozzle head 12 includes a P-axis motor 32 provided on the motor support 35 as the suction / discharge mechanism 23, a ball screw 30 that is rotationally driven by the P-axis motor 32, and the ball screw 30. A drive plate 28 provided with a nut portion 33 to be screwed onto the drive plate, four pistons 26 arranged and supported on the drive plate 28 at the predetermined pitch, and the piston 26 sliding inside thereof. And a cylinder 24. At the lower end of the cylinder 24, the nozzle 22 to which the tip (75, 76, 80) can be attached is provided. A lower end of the ball screw 30 is rotatably supported by the substrate 39.

  The nozzle head 12 is further provided with a detaching mechanism (29, 27) for detaching the tip (75, 76, 80) from the nozzle 22. The desorption mechanism (29, 27) is larger than the outer diameter of the nozzle 22, but is provided at the outer diameter of the opening for mounting the tip (75, 76, 80) or at the upper end of the tip (75, 76, 80). A detaching plate 29 arranged at the predetermined pitch so that the nozzles 22 can simultaneously penetrate four holes (not shown) having an inner diameter smaller than a flange, a protrusion, and the like, and the four The lower end is attached at a position near both ends of the demounting plate 29 along the X-axis direction (row direction) so as to sandwich the hole from both sides, and the upper end is attached to the driving plate 28 so that the demounting is performed. And two support pillars 27 for interlocking the plate 29 with the driving plate 28.

  Therefore, in order to detach the tip (75, 76, 80) from the nozzle 22, the drive plate 28 is moved further downwardly beyond the moving range along the vertical direction of the piston 26 for suction and discharge. The tip (75, 76, 80) is dropped from the nozzle 22 by moving along the axial direction. Therefore, when the tip (75, 76, 80) is used for suction and discharge, the detaching plate 29 is located above the tip (75, 76, 80) with the nozzle 22 passing through the hole. It moves up and down within the movement range.

  The magnetic force means 43 is provided below the substrate 39 of the nozzle head 12. In the magnetic force means 43, six permanent magnets 36 are arranged at the predetermined pitch along the X-axis direction (row direction). It is provided so as to be movable along the Y-axis direction (column direction) so as to approach and separate from each axis of the nozzle 22. The movement of the permanent magnet 36 along the Y-axis direction is executed by a motor 37. Since the number of permanent magnets 36 that is two more than the number of nozzles is arranged and only the four permanent magnets 36 at the center are used, the dispensing tips at both ends are also at the other center. The magnetic field can be applied under the same conditions as the two dispensing tips.

  The nozzle head 12 is provided so as to be movable along the X-axis direction and the Y-axis direction with respect to the stage 14 or the housing 16 by an XY-axis moving mechanism 20. The XY axis moving mechanism 20 includes a head support member 55 provided so as to be movable along the Y axis direction with respect to the casing 16 or the stage 14, and provided on the casing 16. A Y-axis motor 56 for driving a chain gear for moving along the Y-axis direction, a chain gear 58 around which a timing belt is stretched, and the head support member 55 are supported so as to be movable along the Y-axis direction. Therefore, it has two shafts 54 attached to the housing 16 and extending along the Y-axis direction.

The head support member 55 is provided with a rail 60 along the X-axis direction and a slider that is guided by the rail 60 and is slidable on the rail. In order to drive the head 12 along the X-axis direction, it has a timing belt 48 that is stretched over five chain gears 50 and 51, and a motor 52 that rotationally drives the chain gear 51.
In FIG. 1, the rods 88 and 90 of the upper lid closing action mechanism 19 are not shown because they are behind the nozzle 22.

  FIG. 2 shows various chips (75, 76, 77, 80) that can be attached to the nozzle 22.

  FIG. 2 (a) shows the perforating tip 75, which is used only for perforating a prepack cartridge container, which will be described later, and has a sharp tip and a thin film covering the opening of the prepack cartridge container. Can be drilled. A nozzle mounting opening 75a for mounting on the nozzle 22 is provided at the upper end.

  2B shows the PCR chip 80, FIG. 2C shows the genome extraction chip 76, and FIG. 2D shows the analysis chip 77, respectively. The PCR chip 80 and the genome extraction chip 76 correspond to the dispensing chip and have large diameter tubes 80a and 76a, small diameter tubes 80b and 76b, and transition portions 80c and 76c, respectively. Nozzle mounting openings 80e and 76e are provided at the upper ends of the tubes 80a and 76a. The genome extraction chip 76 is suitable for performing separation by adsorbing magnetic particles on the inner wall by applying a magnetic field from the outside to the inside of the small-diameter tube 76b.

  FIG. 2 (d) shows an analysis chip 77 having a large diameter tube 77a provided with a mounting opening 77e and a fitting portion 77c, and a small diameter to which the upper end of the fitting portion 77c is fitted and attached. A plurality of particulate carriers 77d are enclosed in the small-diameter tube 77b, and a predetermined chemical substance is provided on the surface of the particulate carrier 77d so that it can be bonded or can be bonded. Yes. In addition, a predetermined chemical substance that can bind to or bind to each particulate carrier, the arrangement position of the particulate carrier, the shape of the particulate carrier, or the label of the particulate carrier (for example, fluorescent substance, chemiluminescence, dye, etc.) ) In advance.

  Here, the “predetermined chemical substance” refers to a biological substance or the like, for example, a chemical substance including a genetic substance such as a nucleic acid, a biological polymer such as a protein, sugar, sugar chain, or peptide, or a low molecule. The biological material is used for detection, capture, separation, extraction and the like of the binding of the biological material as a receptor having a binding property to the biological material as a ligand. Examples of the receptor include genetic materials such as nucleic acids, genetic materials such as nucleic acids having binding properties to proteins, sugar chains, peptides and the like, and biological materials such as proteins, sugar chains and peptides. In addition, a living body itself such as a cell, a virus, or a plasmid can be used as the biological substance or instead of the biological substance.

  “Binding” includes binding at least one chemical substance to the particulate carrier directly or indirectly through another kind of substance. Examples of bonding include, for example, physical adsorption, hydrogen bonding, and electrical interaction in addition to covalent bonding and chemical adsorption. Alternatively, there are cases where the binding substance possessed by the carrier on the particles and various substances bind with each other by a specific reaction or other methods. In addition, the particulate carrier may be formed of a porous material, an uneven material, or a fibrous material, thereby increasing the ability to react with or bind to various materials.

For binding, for example, the particulate carrier expresses or generates a functional group. For this purpose, for example, the peptide bonds possessed by “polyamide polymers”, such as silk, various nylons, wholly aromatic polyamides such as PPTA (polyparaphenylene terephthalamide), heterocycle-containing aromatic polymers, etc. are hydrolyzed. By decomposing, a functional group used for binding a biological substance is expressed or generated. Examples of the functional group capable of binding to a biological substance include a carboxyl group —COOH, an amino group —NH _{2 and the} like, or a derivative group thereof. Here, the porous diameter suitable for binding of biological substances is, for example, several micrometers or less.

  The particulate carrier 77d is a particulate solid having a size that can be introduced and held in the small-diameter tube 77b of the analysis chip 77. The size of the particulate carrier is, for example, a size of 0.1 mm to several mm in diameter or diameter. In the temperature control region of the analysis chip 77 that holds the particulate carrier 77d, the volume of the space excluding the held particulate carrier 77d is, for example, a volume of several μl to several hundreds of microliters. is there.

  The material of the particulate carrier 77d is a substance that is insoluble in a liquid sample. For example, metal compounds such as metals, semiconductors, metalloids, and metal oxides, ceramics, inorganic substances such as glass and silica, rubber, latex Or a polymer material such as a resin such as polystyrene, polypropylene, polyester, acrylic, cellulose, a fiber material such as nylon as described above, or an organic material such as a natural fiber material.

  FIG. 3 is a plan view showing in detail the stage 14 according to the first embodiment of the present invention from above.

  On the upper left side of the stage 14, a container 74 in which 12 chip accommodating portions are arranged in a matrix of 3 rows × 4 columns and a container 74 in which 16 chip accommodating portions are arranged in a matrix of 4 rows × 4 columns 78, two liquid tanks 72 for storing alcohol, and two containers 70 in which liquid storage portions 71 (for example, 200 μl) capable of storing four specimens are arranged in a line. Yes. The interval between the rows is equal to the predetermined pitch (for example, 18 mm).

  Of the twelve chip storage portions of the container 74, four chip storage portions 74a store four drilling tips 75, and eight chip storage portions 74b store magnetic particles on the inner wall. Eight genome extraction chips 76 serving as the above-mentioned dispensing chips suitable for adsorbing to the gel are accommodated. Similarly, 12 PCR chips 80 serving as the dispensing chips are accommodated in a 3 × 4 chip accommodating portion 78a among the chip accommodating portions of the container 78, and four chip accommodating portions 78b. Is in a vacant state.

  On the lower left side of the stage 14, eight prepack cartridge-like containers 62 and 63 are arranged in four rows at the predetermined pitch. Each of the cartridge-like containers 62 and 63 has a liquid storage part 40a for incubation, a hole part 40b for holding a tube, and a liquid storage part 40c in which ten predetermined reagents are stored in advance. It is covered with a thin film. The cartridge container 62 is a genome extraction cartridge container that contains a reagent for genome extraction, for example, magnetic particles coated with silica or the like, a separation solution, a buffer solution, a washing solution, and the like. The container 63 is a cartridge container for analysis in which a reagent for analysis and the like, for example, a reagent such as a nucleotide having a predetermined base sequence labeled with a fluorescent substance, various buffer solutions, various washing solutions and the like are accommodated. A heater 68 is provided in the stage 14 for heating the tube held in the incubation liquid container 40a or the tube holding hole 40b.

  Further, on the lower right side of the stage 14, a PCR reagent cooling block 64 in which twelve liquid storage portions are arranged in a matrix of 3 rows × 4 columns (for example, to cool at 4 ° C.), and A container 44 composed of four chip accommodating portions 42 arranged in a line for accommodating the analysis chips 77 in which the plurality of particulate carriers 77d are enclosed is provided in a small-diameter tube of a dispensing chip. . Of the 12 liquid storage sections of the PCR reagent cooling block 64, the 8 liquid storage sections 64a include PCR reagents such as primers, polymerases, reverse primers, bases, etc. A small amount (for example, several μl) is used in the liquid container. Therefore, each of the liquid storage portions 64a of the PCR reagent cooling block 64 has a capacity of 200 μl so that different reagents can be stored together for easy handling. Four of the liquid storage parts of the PCR reagent cooling block 64 are the extracted genome storage liquid storage parts 64b, which have a capacity of 1.5 ml and have one PCR chip 80 in the nozzle head 12. Use with. Note that the row spacing of each column of these containers is equal to the predetermined pitch.

  Further, on the upper right side of the stage 14, 32 reaction vessels 98 (FIGS. 5, 6, 16, etc.) arranged in a matrix of 8 columns × 4 rows that can be temperature-controlled by the temperature controller 100. And a top-covered reaction vessel 82 that covers the opening so as to be openable and closable in the upward direction. The pitch of the reaction vessel 98 is set to half the predetermined pitch (for example, 9 mm pitch). As a result, temperature control can be performed in an integrated state. The reaction vessel 82 with an upper lid is further provided with a fixing block 87 fixed to the stage 14, an opening / closing hinge 87a (see FIG. 4) provided on the fixing block 87 and driven to open and close according to instructions. An upper lid support frame 84 as an upper lid support member that is pivotally supported by the fixing block 87 via an opening / closing hinge 87a and provided with a through hole 83 that loosely fits and elastically supports the upper lid 86. .

  Here, the fixing block 87 and the opening / closing hinge 87a correspond to the shaft support portion, and belong to the reaction vessel 82 with an upper lid together with an opening / closing driving motor (see reference numeral 201 in FIG. 10) described later. Note that the size of the stage 14 described above is, for example, about 0.5 m to 1 m in length and width.

  4 shows an enlarged plan view (FIG. 4A) and an enlarged back view (FIG. 4B) of the nozzle head 12 shown in FIG. Note that the same reference numerals as those in FIG.

  As shown in FIGS. 4A and 4B, a rod 90 is further provided on the driving plate 28 provided with a nut portion 33 that is rotationally driven by the P-axis motor 32 via the ball screw 30. Are linked. A rod 88 is further connected to the cylinder support 25 provided with a nut portion 41 that is rotationally driven by the Z-axis motor 34 via the ball screw 31. That is, the rod 90 is driven by the suction / discharge mechanism 23 (see FIG. 1), and the rod 88 is driven by the moving mechanism 21 (see FIG. 1).

Grooves 94 and 92 as recesses are provided at the lower ends of the rod 90 and the rod 88. The hook 85 provided on the upper lid 86 is provided with a U-shaped notch 85a cut into a size that can be fitted into the grooves 92 and 94, and is engaged with the moving mechanism (20, 21). Is possible. When the upper cover 86 is pressed, shaken or moved using the rods 88, 90, the height position is controlled so that the lower ends of the rod 90 and the rod 88 are maintained at the same height level. The lower ends of the ball screws 30 and 31 are pivotally supported on the substrate 39 by ball bearings 30a and 31a. The nozzle head 12 is incorporated in a frame 96 whose front and rear surfaces are open.
Here, the rods 88 and 90, the grooves 92 and 94, the moving mechanisms (20 and 21), the suction / discharge mechanism 23, the hook 85, and the like are provided as an upper lid that can be opened and closed with respect to the opening of the reaction vessel 82 with an upper lid. It corresponds to the action mechanism 19 at the time of closing the upper lid that enables 86 to be pressed, shaken or moved.

FIG. 5 shows in detail the state before the reaction vessel 82 with an upper lid is pressed.
As shown in the plan view of FIG. 5A, a hook 85 is provided on the upper pressed surface 86a when the opening of the reaction vessel 98 is closed by the upper lid 86 of the reaction vessel 82 with an upper lid, The hook 85 has a plate 85b provided parallel to a predetermined height from the pressed surface 86a, and the plate 85b includes the grooves 94 and 92 as the recesses provided at the lower ends of the rods 88 and 90. The two U-shaped cutout portions 85a that can be fitted are provided, and the edge portion of the plate 85b opposite to the side on which the U-shaped cutout portion 85a is provided is bent so that the end thereof is covered with the cover. It is attached to the pressing surface 86a.

  FIG. 5B is a cross-sectional view taken along line AA of FIG. 5A, and the upper lid 86 faces the pressed surface 86a pressed by the rods 88 and 90 and the pressed surface 86a. And has a covering surface 86b that covers the opening of the reaction vessel 98, and a side surface 86c that connects the pressed surface 86a and the covering surface 86b. Is formed.

  Further, the side surface 86c of the upper lid 86 extends beyond the covering surface 86b so as to surround the upper side of the reaction vessel 98 when the opening is closed, and forms an edge wall portion 107. A spring support protrusion 108 is provided protruding outward in the linear direction.

  In the upper lid support frame 84, the covering surface 86b can cover the opening of the reaction vessel 98, the pressed surface 86a of the upper lid 86 is exposed upward, and the covering surface 86b is exposed downward. Thus, a through hole 83 through which the main body of the upper lid 86 passes is provided. One end of a spring 102 as an elastic member is attached to the upper lid support frame 84, and the other end is attached to the spring support protrusion 108 of the upper lid 86. The upper lid 86 is located in the through hole 83. The spring 102 is urged and supported in the direction from the covering surface 86b toward the pressed surface 86a. Therefore, in a state where the pressed surface 86 a is not pressed by the rods 88, 90, the covering surface 86 b or a pad described later only covers the opening of the reaction vessel 98 and is not in contact. Here, in addition to the upper lid support frame 84, the through hole 83, the spring 102, and the spring support projection 108 correspond to the upper lid support member.

  The upper lid support frame 84 is pivotally supported at one end by the fixing block 87 via the opening / closing hinge 87a. The opening / closing hinge 87a is opened / closed by an instruction by an opening / closing driving motor (not shown) provided on the stage 14 or the fixing block 87.

  A pad 106 is provided so as to be in close contact with the covering surface 86 b and the edge wall portion 107, thereby enhancing the sealing performance of the opening of the reaction vessel 98. The hollow portion 105 is preferably provided with a heating panel to prevent condensation at the opening of the reaction vessel 98.

  Furthermore, each reaction vessel 98 is provided with a protrusion 103 along the outer peripheral surface thereof, and a plurality of (this example) having an inner diameter smaller than the outer diameter of the protrusion 103 and larger than the outer diameter of each reaction container 98 main body. In this case, the reaction vessel 98 is brought into close contact with the covering surface 86b or the pad 106 by attaching the lifting prevention plate 104 of each reaction vessel 98 having 32 holes) to the temperature controller 100. The situation where it is lifted is prevented.

  The reaction vessel 82 with an upper lid is provided with a temperature controller 100 so as to surround the lower side of the reaction vessel 98 to control the temperature of the reaction vessel 98. The temperature controller 100 is provided with a thermal cycler having a Peltier element that can raise and lower the temperature according to an instruction.

FIG. 6 shows a state when the reaction vessel 82 with the top lid is pressed, and the groove 92 of the rod 88 and the groove 94 of the rod 90 are formed in the two U-shaped notches 85 a of the hook 85. While fitting each, the rods 88 and 90 are moved down to press the pressed surface 86a when the opening is closed by the upper lid 86, so that the covering surface 86b is interposed through the pad 106. The state which pressed and sealed the opening part of the said reaction container 98 is shown.
Here, the hook 85, the U-shaped notch 85a, etc., together with the rods 88, 90, correspond to the action mechanism 19 when the upper lid is closed.

  After the temperature control, when the upper lid 86 is opened, the rods 88 and 90 are moved up and down or left and right with respect to the reaction vessel 98 using the suction / discharge mechanism 23 and the moving mechanisms 20 and 21. The sticking between the covering surface 86b or the pad 106 of the upper lid 86 and the opening of the reaction vessel 98 can be eliminated.

  Based on FIG. 7 thru | or FIG. 9, operation | movement in the case of performing PCR processing by temperature-controlling DNA or a genome using the said temperature control reaction processing apparatus 10 which concerns on the 1st Embodiment of this invention is demonstrated. .

  In step S1, for example, samples of oral mucosa, blood, nails and the like are collected from four subjects and put into water and stored in each liquid storage portion 71 of the container 70. After the tip for punching 75 is attached to the nozzle 22 by simultaneously lowering the nozzles 22 in the tip accommodating portion 74a to perforate a thin film covering the opening of the cartridge-like container 62, the attachment / detachment mechanism The genome extraction chip 76, which is detached from the chip accommodating portion 74a and accommodated in the chip accommodating portion 74b, is attached to the nozzle 22 by lowering the nozzle 22. The four genome extraction chips 76 are moved by the moving mechanisms 20, 21, and the necessary reagents are simultaneously collected from the liquid storage unit 40 c storing the corresponding extraction reagents using the suction / discharge mechanism 23. Aspirate, discharge together with a predetermined reagent containing magnetic particles in the liquid container 71 containing the specimen, and agitate and incubate by repeating aspiration and discharge all at once by the aspiration and discharge mechanism 23, and react to react. DNA is bound and captured on the surface of the magnetic particle.

  In step S2, the magnetic means 43 is used to move the permanent magnet 36 to a large diameter tube 76a, a small diameter tube 76b formed narrower than the large diameter tube 76a, and a transition connecting the large diameter tube 76a and the small diameter tube 76b. The DNA is separated by bringing a magnetic field into contact with the thin tubes 76b of the four genome extraction chips 76 comprising the portions 76c all at once, and adsorbing the magnetic particles on the inner walls of the thin tubes 76b.

  In step S3, the four genome extraction chips 76 are moved by the moving mechanism (20, 21) while adsorbing the magnetic particles capturing the DNA on the inner wall, and the detachment liquid is accommodated. By positioning the liquid container 40c of the cartridge-like container 62, inserting the tip opening 76d of the genome extraction chip 76 into the container 40c, and repeating suction and discharge while adsorbing the magnetic particles to the inner wall. The DNA is separated from the magnetic particles. The DNA solution containing the DNA separated from the magnetic particles is discharged into the four genome storage liquid storage portions 64b of the PCR reagent cooling block 64 and stored, and then the genome extraction chip 76 is mounted. The chip accommodating portion 74b that is vacant while adsorbed to the magnetic particles on the inner wall is detached.

  In step S4, the nozzle head 12 is moved, and the nozzles 22 arranged at the end of the nozzle head 12 are moved to the tip accommodating portion 78a at the end of the container 78, and then the Z-axis moving mechanism 21 is moved. Thus, the nozzle 22 is lowered to insert the nozzle 22 into the nozzle mounting opening 80e of the single PCR chip 80 accommodated.

  In step S5, the opening / closing hinge 87a is driven to open and close as shown in FIG. 7, thereby opening the upper lid 86 upward and exposing the opening of the reaction vessel 98 to the outside. Next, using the single PCR chip 80, each reaction container 98 (individually 9 mm, half of the predetermined pitch) from the corresponding one liquid storage section 64a of the PCR reagent cooling block 64 is used. Pitch in the reaction vessel 82 with the top lid by aspirating a PCR reagent, for example, the primer-containing solution, which is multiple times (in this example, 4 times) of the amount required for one (eg, several μl) Are accommodated by sequentially dispensing into a plurality of reaction vessels 98 of different sizes. At that time, since the amount of liquid multiple times the amount required for one reaction vessel is handled, the amount of liquid increases and it is easy to perform suction and discharge. The above steps are repeated until the necessary reagents are dispensed.

  In step S6, the nozzle head 12 is detached from the empty chip housing portion 78b of the container 78 after removing one PCR chip 80 attached to the nozzle head 12 using the moving mechanisms 20 and 21. A new PCR chip 80 housed in the second chip housing portion 78a of the container 78 is attached to the end of the nozzle head 12 or the nozzle 22 arranged second, and the moving mechanism 20 , 21 is used to move to the genome storage solution storage section 64b storing the extracted DNA of the PCR reagent cooling block 64, and the corresponding of the DNA extracted from four patients A plurality of times (in this example, 4 times) the amount of DNA solution required in each reaction container 98 is aspirated, moved to the reaction container 98, and sequentially dispensed into the required number of containers. The above process is repeated for all four patients.

  In step S7, after attaching the floating prevention plate 104 to the reaction vessel 98 with respect to the reaction vessel 82 with an upper lid, a pad 106 is attached in close contact with the covering surface 86b of the upper lid 86, and the open / close hinge The upper lid support frame 84 is rotated by rotationally driving 87 a so that the opening of the reaction vessel 98 is covered with the covering surface 86 b of the upper lid 86 or the pad 106.

  In step S8, the nozzle head 12 is moved by the moving mechanisms 20 and 21 to the empty chip housing portion 78b, and then the PCR chip 80 is removed from the nozzle 22 by the detaching mechanism (29, 29). 27).

  In step S9, as shown in FIG. 8, the rod 88 and the rod 90 are interlocked with the nozzle 22 using the suction / discharge mechanism 23 and the moving mechanism (20, 21), or together with the piston in the cylinder. In conjunction with this, after being lowered to a predetermined height with respect to the stage 14, it is moved along the X-axis direction by the XY-axis moving mechanism 20 to the reaction vessel 82 with an upper lid where the upper lid 86 is located. Thereafter, the XY axis moving mechanism 20 is used to move along the Y axis direction.

  Here, the “predetermined height” is a height at which the grooves 92 and 94 formed at the lower ends of the rods 88 and 90 can be engaged with the hook 85 of the upper lid 86.

  In step S10, as shown in FIG. 9, the groove 88 of the rod 88 and the groove 94 of the rod 90 are fitted into the U-shaped cutout portions 85a of the hook 85 of the upper lid 86, whereby the rod 88, 90 and the upper lid 86 are engaged with each other via the hook 85 and connected.

,
In step S11, the rod 88 and the rod 90 are further moved downward to press the pressed surface 86a of the upper lid 86, thereby bringing the covering surface 86b and the pad 106 into the reaction vessel 98. The opening will be sealed.

  In step S12, the temperature control according to the PCR method is performed on the reaction vessel 98 by the temperature controller 100. In the temperature control according to the PCR method, the temperature of the reaction vessel 98 is set to 94 ° C. in order to denature the double-stranded sample DNA that has been input into single-stranded DNA, and then single-stranded DNA. In order to carry out annealing or hybridization between the primer and the primer, the temperature of the reaction vessel 98 is set to 50 ° C. to 60 ° C. Next, in order to synthesize a DNA strand complementary to the single strand, the operation of setting the temperature to 74 ° C. and incubating is performed as one cycle, and is repeated a predetermined number of times, for example, temperature control is performed for about several minutes. I do.

  In step S13, after temperature control is completed, the rods 88 and 90 are shaken or moved in the vertical direction or the horizontal direction (X-axis direction) using the suction / discharge mechanism 23 or the moving mechanism (20, 21). By repeating the removal of moisture due to condensation on the covering surface 86b or the pad 106 of the upper lid 86, or by shifting the position, the opening of the reaction vessel 98 adheres to the pad 106 or the covering surface 86b. The upper lid 86 is easily opened by releasing the state.

  In step S14, the upper lid 86 is opened, the PCR products accommodated in each reaction vessel 98 are collected, and various inspections and analyzes using the analysis chip 77 and the like are performed using the PCR products. Will be done.

FIG. 10 shows a temperature controlled reaction processing apparatus 110 according to the second embodiment.
The temperature control reaction processing apparatus 110 includes a suction / discharge mechanism 123 (cylinder 124 or the like) that performs gas suction / discharge, and the various chips (76, 80, FIG. 2), a nozzle head 112 having two or more nozzles 122 (six in this example) arranged in a predetermined pitch, a stage 114 provided with various containers, and the nozzle 122. A moving mechanism (120, 121) for moving the stage 114; The moving mechanism includes a Y-axis moving mechanism 120 mainly provided outside the nozzle head 112 and a Z-axis moving mechanism 121 serving as the up-and-down moving mechanism provided in the nozzle head 112.

  The nozzle head 112, the moving mechanism (120, 121), and the stage 114 are incorporated in a housing 116 whose front surface and one side surface are open. An operation panel 118 is provided on the other side surface of the casing 116. The operation panel 118 includes a display and an operation keyboard, and information provided in the casing 116 together with the nozzle head, a moving mechanism, a suction / discharge mechanism, a temperature controller (described later) provided on the stage 114, and the like. It is electrically connected to a processing device (not shown). As described in the first embodiment, the information processing apparatus includes a peripheral device including an external memory such as a CD or DVD driver in addition to a CPU and a memory, and the nozzle head 112 is included in the memory. A program for controlling the suction / discharge mechanism 123, the moving mechanism (120, 121), and therefore the upper lid closing action mechanism 119 described later, the temperature controller 200 described later, and the like are introduced.

  The nozzle head 112 includes a Z-axis motor 134 mounted on a motor support 135, a ball screw 131 that is rotationally driven by the Z-axis motor 134, and a nut portion 141 that is screwed with the ball screw 131. And a cylinder support 125 that fixes and supports the six cylinders 124 arranged at the predetermined pitch, and moves the nozzle 122 in the Z-axis direction (vertical direction). This corresponds to 121. The lower end of the ball screw 131 is rotatably supported on the substrate 139.

  Further, the nozzle head 112 includes a P-axis motor 132 provided on the motor support 135 as the suction / discharge mechanism 123, a ball screw 130 that is rotationally driven by the P-axis motor 132, and the ball screw 130. A drive plate 128 provided with a nut portion 133 to be screwed onto the drive plate, six pistons 126 arranged and supported on the drive plate 128 at the predetermined pitch, and the piston 126 sliding inside thereof. And a cylinder 124. At the lower end of the cylinder 124, the nozzle 122 to which the tip (76, 80) can be attached is provided. A lower end of the ball screw 130 is rotatably supported by the substrate 139 1.

  The nozzle head 112 is further provided with a detaching mechanism (129, 127) for detaching the tip (76, 80) from the nozzle 122. The desorption mechanism (129, 127) is larger than the outer diameter of the nozzle 122, but has an outer diameter of the opening for mounting the tip (76, 80) or a flange or protrusion provided at the upper end of the tip (76, 80). 6 holes (not shown) having an inner diameter smaller than a strip or the like are arranged at the predetermined pitch so that the nozzles 122 can pass through, and the 6 holes are provided on both sides. The lower end is attached at a position near both ends of the detachable plate 129 along the X-axis direction so as to be sandwiched between the upper and lower ends, and the upper end is attached to the drive plate 128 to attach the detachable plate 129 to the drive plate. And two support pillars 127 interlocked with 128.

  Therefore, in order to detach the tip (76, 80) from the nozzle 122, the drive plate 128 is moved further down the Z axis direction beyond the moving range along the vertical direction of the piston 126 for suction and discharge. The tip (76, 80) is peeled off from the nozzle 122 by moving along the nozzle. Therefore, when the tip (76, 80) is used for suction and discharge, the detaching plate 129 is moved in the movement range above the tip (76, 80) with the nozzle 122 passing through the hole. It will move up and down.

  The magnetic force means 143 is provided below the substrate 139 of the nozzle head 112. In the magnetic force means 143, eight permanent magnets 136 are arranged at the predetermined pitch along the X-axis direction (row direction), and the six permanent magnets 136 at the center excluding both ends of the eight permanent magnets 136 are arranged as described above. It is provided so as to be movable along the Y-axis direction (column direction) so as to approach and separate from each axis of the nozzle 122. The movement of the permanent magnet 136 along the Y-axis direction is executed by the motor 137. Since the number of permanent magnets 136, which is two more than the number of nozzles, is arranged and only the six permanent magnets 136 in the center are used, the dispensing tips in the center of the dispensing tips at both ends are also used. The magnetic field can be exerted under the same conditions.

  The nozzle head 112 is provided so as to be movable only along the Y-axis direction with respect to the stage 114 or the housing 116 by the Y-axis moving mechanism 120. The Y-axis moving mechanism 120 includes a head support member 155 provided so as to be movable along the Y-axis direction with respect to the casing 116 or the stage 114, and attached to the casing 116 along the Y-axis direction. Two shafts 154 extending.

  The nozzle head 112 includes a driving plate 128 provided with a nut portion 133 that is rotationally driven by the ball screw 130, and a cylinder provided with a nut portion 141 (not shown) that is rotationally driven by the ball screw 131. And a support body 125. The second embodiment differs from the first embodiment in that the upper lid closing action mechanism 119 does not have the rod 90 and the rod 88.

  Instead, the nozzle 122 is used to press, shake, or move the upper lid. The contents of the action of the upper lid closing action mechanism 119 are controlled based on the contents of the instruction and the like as described in the first embodiment. The height position of the nozzle 122 is controlled so that the tip of the nozzle 122 has a height level that can be fitted into the U-shaped notch 185a of the hook 185. The lower ends of the ball screws 130 and 131 are pivotally supported on the substrate 139 by ball bearings (131a). The upper lid closing action mechanism 119 includes a nozzle 122, a moving mechanism (120, 121), a hook 185, and the like. The nozzle head 112 is incorporated in a frame body 196 whose front and rear surfaces are open. Therefore, since the size of the motor 137 for driving the magnetic force means 143 is not limited by the presence of the rods 90 and 88, an effect is obtained that a sufficiently large motor can be used for the magnetic force means 143.

  11 (a) and 11 (b) are plan views showing the stage 114 according to the second embodiment of the present invention in detail from the upper side. FIG. 11 (a) shows an upper lid 186, which will be described later, as a reaction vessel 198. FIG. 11 (b) shows a state in which the upper lid 186 is closed with respect to the opening of the reaction vessel 198. Since FIG. 11B shows the same thing as FIG. 11A, the code | symbol was abbreviate | omitted except the case where it was hard to understand about the same thing as FIG. 11A.

  The stage 114 is sandwiched between six liquid storage parts 174a for collecting the extracted genome, six liquid storage parts 174d for storing specimens, and the liquid storage parts 174a and 174d. A container 174 having chip receiving portions 174b and 174c arranged in a matrix of 12 4 rows x 6 columns provided in the above, and two types of PCR reagents, for example, primers, polymerase, reverse primer, bases, etc. And a PCR reagent cold block 170 provided in a matrix of 2 rows × 6 columns composed of liquid storage portions 170a and 170b that can store 2 types selected from 1 row by row. The interval between the rows is equal to the predetermined pitch (for example, 18 mm).

  In the chip accommodating portions 174b and 174c of the container 174, six genome extraction chips 76 as the dispensing chips suitable for adsorbing magnetic particles on the inner wall and six PCRs as the dispensing chips are provided. Each chip 80 is accommodated. Unlike the first embodiment, the cartridge-like container 162 is pierced by a piercing needle (not shown) provided in the nozzle head 112.

  The stage 114 is provided with six pre-packed cartridge-like containers 162 arranged along the row direction at intervals of the predetermined pitch. Each cartridge-like container 162 has a liquid storage part 140a for incubation, a hole part 140b for holding a tube, and 10 liquid storage parts 140c in which 10 predetermined reagents are stored in advance. Covered with a thin film. The cartridge-like container 162 is a genome-like cartridge-like container in which each of the reagents for genome extraction, for example, magnetic particles coated with silica or the like, separation liquid, buffer liquid, washing liquid, and the like are accommodated. In the stage 114, there is provided a heater capable of heating the tube held in the incubation liquid container 140a or the tube holding hole 140b.

  Further, the stage 114 has twelve reaction vessels 198 (the pitch is the same as the predetermined pitch, 18 mm in this example) arranged in a matrix of 2 rows × 6 columns capable of controlling the temperature, A reaction vessel 182 with an upper lid is provided that has an upper lid 186 that covers the opening so as to be openable and closable. The reaction vessel 182 with an upper lid is further provided with a fixing block 187 provided on the stage 114, an opening / closing hinge 187a provided on the fixing block 187 and driven to open and close according to instructions, and the fixing via the opening / closing hinge 187a. And an upper lid support frame 184 as the upper lid support member provided with a through hole 183 that is pivotally supported by the block 187 and elastically supports the upper lid 186 by being loosely fitted thereto.

  Further, one end of a spring 202 as an elastic body is attached to the upper lid support frame body 184, and the other end is attached to a spring support projection 208 of the upper lid 186, and the upper lid 186 is disposed in the through hole 183. In this case, the spring 202 is urged and supported in the direction from the covering surface 186b toward the pressed surface 186a. Therefore, in a state where the pressed surface 186 a is not pressed by the nozzle 122, the covering surface 186 b or a pad 206 described later only covers the opening of the reaction vessel 98 and is not in contact. Here, in addition to the upper lid support frame body 184, the through hole 183, the spring 202, and the spring support projection 208 correspond to the upper lid support member.

  The upper lid support frame 184 is pivotally supported at one end by a fixing block via the opening / closing hinge 187a. The opening / closing hinge 187a is driven to open / close by an instruction by an opening / closing motor (not shown) provided on the stage 114 or the fixing block. The stage 114 is, for example, about 0.5 to 1 m in length and width.

  Based on FIGS. 12 to 15, when the temperature-controlled reaction processing apparatus 110 according to the second embodiment of the present invention is used, the DNA or genome of the corresponding gene is subjected to temperature control to perform PCR processing. The operation will be described.

  In step S21, for example, samples of oral mucosa, blood, nails, and the like were collected from six subjects, and poured into water, stored in the liquid storage unit 171 and provided in the nozzle head 112. After a thin film covering the opening of the liquid container 140c in which the corresponding reagent is stored in the cartridge-like container 162 is punched by a needle for punching (not shown), the moving mechanism (120, 121) is used. Then, the genome extraction chip 76 is moved to the chip storage portion 174b in which the genome extraction chip 76 is stored, and the nozzles 122 of the nozzle head 112 are moved down, so that the six genome extraction chips 76 are connected to the six nozzles 122. After being attached all at once, the liquid is moved by the moving mechanism (120, 121), and the liquid collection container in which the corresponding extraction reagent is stored using the suction / discharge mechanism 123. Necessary reagents are aspirated from the part 140c, discharged together with a predetermined reagent containing magnetic particles into the liquid storage part 171 containing the specimen, and stirred and incubated by repeating suction and discharge by the suction and discharge mechanism. By reacting, DNA is bound to the surface of the magnetic particle and captured.

  In step S22, the magnetic means 143 is used to move the permanent magnet 136 to the large diameter tube 76a, the small diameter tube 76b formed narrower than the large diameter tube 76a, and the transition connecting the large diameter tube 76a and the small diameter tube 76b. The magnetic particles are separated by adsorbing the magnetic particles on the inner wall of the small-diameter tube 76b by applying a magnetic field by simultaneously approaching the small-diameter tubes 76b of the six genome extraction chips 76 composed of the portions 76c.

  In step S23, the six genome extraction chips 76 are moved by the moving mechanism (120, 121) while the magnetic particles capturing the DNA are adsorbed on the inner wall, and the liquid storage unit stores the divergence liquid. It is positioned in the portion 140c, the tip port portion 76d of the genome extraction chip 76 is inserted into the liquid storage portion 140c, and the suction and discharge are repeated while the magnetic particles are adsorbed on the inner wall. Deviate. After discharging the DNA solution containing DNA deviated from the magnetic particles into the liquid container 174a of the container 174, the six genomic extraction chips 76 are held while adsorbing the magnetic particles on the inner wall. The original six chip accommodating portions 174b are desorbed all at once.

  In step S24, the nozzle head 112 is moved so that the nozzle 122 is positioned on the chip accommodating portion 174c in which the PCR chip 80 is accommodated, and then the six axes are moved by the Z-axis moving mechanism. By simultaneously lowering the nozzles 122, the nozzles 122 are simultaneously inserted into the nozzle mounting openings 80e of the six PCR chips 80 and mounted.

  In step S25, as shown in FIG. 12, the opening / closing hinge 187a is driven to open and close by rotating the motor 201 to open the upper lid 186 upward, and the opening of the reaction vessel 198 is exposed to the outside. deep. Next, the six PCR chips 80 are used to move to the PCR reagent cooling block 170, and, for example, the primer-containing liquid is simultaneously mixed from the liquid storage part 170a in which the PCR reagent is stored. And are discharged together into the six reaction vessels 198 using the moving mechanism (120, 121). Similarly, the PCR reagent is also sucked into the liquid container 170b and discharged into the six reaction vessels 198.

  In step S26, the nozzle head 112 is moved to the liquid container 174a containing the extracted DNA or genome in the container 174 using the moving mechanism, and the DNA extracted from six patients is extracted. Alternatively, the genome is sucked all at once into the six PCR chips 80, moved again into the six reaction vessels 198, and discharged and accommodated all at once.

  In step S27, the nozzle head 112 is moved by the moving mechanism to the position of the chip accommodating portion 174c, and then the PCR chip 80 is detached from the nozzle 122 by the detaching mechanism. FIG. 13 shows a state where the PCR chip 80 is detached.

  In step S28, as shown in FIG. 14, the reaction container 182 with the upper lid is attached with the pad 206 in intimate contact with the covering surface of the upper lid 186 after the lifting prevention plate 204 is attached to the reaction container 198. Thereafter, the upper lid support frame 184 is rotated by a predetermined angle by rotationally driving the opening / closing hinge 187a, so that the opening of the reaction vessel 198 is covered with the covering surface 186b of the upper lid 186 or the pad 206. . The reaction vessel 198 is provided with a protrusion 203 along the outer peripheral surface thereof, and a plurality of (this example) having an inner diameter smaller than the outer diameter of the protrusion 203 and larger than the outer diameter of the main body of each reaction vessel 198. In this case, the reaction vessel 198 is brought into close contact with the covering surface 186b or the pad 206 by attaching the floating prevention plate 204 of each reaction vessel 198 having 12 holes) to the upper side of the temperature controller 200. The situation where it is lifted upward is prevented.

  In step S29, as shown in FIG. 14, the position of the nozzle 122 of the nozzle head 112 is lowered to a predetermined height position with respect to the stage 114 using the moving mechanism (120, 121), and then Y The shaft moving mechanism 120 is moved to the position of the upper lid 186.

  Here, the predetermined height is a height at which the lower end portion of the nozzle 122 can be fitted into the U-shaped cutout portion 185 a of the hook 185 of the upper lid 186.

  In step S30, as shown in FIG. 15, the lower end of the nozzle 122 is fitted into the U-shaped notch 185a of the hook 185 of the upper lid 186 so that the upper lid is interposed via the nozzle 122 and the hook 185. Combine with 186.

  In step S31, the nozzle 122 is moved further downward to press the pressed surface 186a of the upper lid 186, thereby sealing the covering surface 186b and the pad 206 with the opening of the reaction vessel 198. Will do.

  In step S32, the temperature control according to the PCR method is performed on the reaction vessel 198 by the temperature controller 200. The temperature control according to the PCR method is such that the temperature of the reaction vessel 198 is set to 94 ° C. in order to denature the input double-stranded sample DNA into single-stranded DNA, and then the single-stranded DNA and In order to perform annealing or hybridization with the primary, the temperature of the reaction vessel 198 is set to 50 ° C. to 60 ° C. Next, in order to synthesize a DNA strand complementary to the single strand, the operation of setting the temperature to 74 ° C. and incubating is performed as one cycle, and is repeated a predetermined number of times, for example, temperature control is performed for about several minutes. I do.

  In step S33, after temperature control is completed, the nozzle 122 is shaken or moved repeatedly in the vertical direction and the horizontal direction (Y-axis direction) using the moving mechanism (120, 121). By releasing the state in which the opening is in close contact with the pad 206 or the covering surface 186b, the upper lid 186 is easily opened.

  In step S34, the upper lid 186 is opened, and the PCR products accommodated in each reaction vessel 198 are collected. Using the PCR products, for example, various tests using the analysis chip 77 or the like can be performed. Analysis will be performed.

  Subsequently, a temperature controlled reaction processing apparatus 210 according to a third embodiment of the present invention will be described with reference to FIGS.

  As shown in FIG. 16, the temperature controlled reaction processing apparatus 210 is replaced with a reaction container 82 with an upper lid having an upper lid 86 that opens upward in the temperature controlled reaction processing apparatus 10 according to the first embodiment. A reaction vessel 282 with an upper lid having an upper lid 286 that opens in a horizontal plane is provided, and other configurations are the same as those of the temperature-controlled reaction processing apparatus 10 according to the first embodiment. Therefore, those having the same reference numerals as those used in the temperature-controlled reaction processing apparatus 10 according to the first embodiment represent the same elements, and thus description thereof is omitted. The upper lid closing mechanism is the same as that according to the first embodiment, but the contents of the control may differ depending on the structure of the reaction vessel with the upper lid.

  As shown in FIGS. 16 and 17A, the reaction vessel 282 with an upper lid according to the present embodiment is provided on the upper right side of the stage 214 and is arranged in a matrix of 8 columns × 4 rows capable of temperature control. 32 reaction vessels 98 (see FIGS. 5 and 6, etc.), a temperature controller 300 provided so as to surround the lower side of the reaction vessel 98 and controlling the temperature of the reaction vessel 98, and its opening An upper lid 286 that can be opened and closed within a horizontal plane, and an upper lid support handle 283 as an upper lid support member for elastically supporting the upper lid 286. A hook 85 is provided on the upper pressed surface 286a when the opening of the reaction vessel 98 is closed by the upper lid 286. The pitch of the reaction vessel 98 is set to half the predetermined pitch (for example, 9 mm pitch). As a result, temperature control can be performed in an integrated state.

  As shown in FIGS. 17B and 17C, the reaction vessel 282 with an upper lid further has an upper lid rotation shaft 284 that protrudes downward from the lower side of the upper lid support handle 283, A plurality of teeth, claws, protrusions, ridges, or grooves are formed on the upper end of the upper lid rotating shaft 284 having a radius larger than that of the upper lid rotating shaft 284 and arranged at one or a certain interval. The lower end portion 284a is provided.

  A coiled spring 302 is provided so as to be coaxially surrounded along the outer periphery of the upper lid rotating shaft 284. The lower end portion 284a of the upper lid rotating shaft 284 is driven by a motor fixed to the stage 214, and is inserted into a hollow rotating cylinder 287 corresponding to the shaft support portion. The coil spring 302 urges the rotating cylinder 287 and the upper lid support handle 283 to axially stretch so that the upper lid 286 or the pad 306 does not contact the opening of each reaction vessel 98. So that it is lifted.

  On the surface facing the lower end portion 284a on the lower side of the inner wall at the upper end inside the rotating cylinder 287, teeth, claws, protrusions, and protrusions that can be engaged with the teeth, claws, protrusions, protrusions, or grooves. Alternatively, when a groove is formed and the lower end portion 284a is in contact with the lower side of the inner wall by the coil spring 302, rotation of the rotating cylinder 287 causes the teeth, claws, protrusions, protrusions, or grooves to The rotation of the opening / closing drive motor (not shown) is transmitted to the upper lid 286 via the rotating cylinder 287, the upper lid rotating shaft 284, etc. The upper lid 286 can be rotated in a horizontal plane without contact.

  Here, reference numeral 304 is a plate for preventing the reaction vessel 98 from being lifted up and attached to the upper side of the temperature controller 300, and reference numeral 306 slides in close contact with the covering surface 286b of the upper lid. It is the pad formed in the cross-sectional L-shape inserted removably. This enhances the hermeticity of the opening of the reaction vessel 98. The hollow portion 305 is preferably provided with a heating panel to prevent condensation at the opening of the reaction vessel 98.

  The floating prevention plate 304 has 32 holes, so that each reaction vessel 98 is prevented from being lifted upward in close contact with the covering surface 286b or the pad 306. Here, in addition to the upper lid support handle 283, the coiled spring 302, the rotating cylinder 287, and the like correspond to the upper lid support member.

  In addition, a temperature controller 300 is provided in the reaction vessel 282 with an upper lid so as to surround the lower side of the reaction vessel 98 to control the temperature of the reaction vessel 98. The temperature controller 300 is provided with a thermal cycler having a Peltier element that can raise and lower the temperature according to an instruction.

  18 (a), 18 (b) and 18 (c), the groove 92 of the rod 88 and the groove 94 of the rod 90 are fitted into the two U-shaped notches 85a of the hook 85, respectively. The rods 88 and 90 are moved down to indicate when the opening is closed by the upper lid 86. By pressing the pressed surface 286a, the covering surface 286b is pressed against the opening of the reaction vessel 98 through the pad 306 and sealed.

  In this case, the lower side of the upper inner wall of the rotating cylinder 287 is separated from the upper side of the lower end portion 284a of the upper lid rotating shaft 284, and the rotation of the opening / closing driving motor is not transmitted to the upper lid 286. Yes.

  Based on FIG. 19 thru | or FIG. 21, operation | movement in the case of performing PCR processing by temperature-controlling DNA or a genome using the said temperature control reaction processing apparatus 210 which concerns on the 3rd Embodiment of this invention is demonstrated. .

  This operation is substantially the same as that in the case of performing the PCR process by controlling the temperature of DNA or genome using the temperature controlled reaction processing apparatus 10 according to the first embodiment described above. Only the processing relating to the reaction vessel 282 with an upper lid is different. Therefore, only the processes related to steps S′5 to S′14 corresponding to steps S5 to S14 related to the processes related to the reaction container 282 with the upper lid will be described.

  In step S′5, as shown in FIG. 19, by rotating the rotary cylinder 287, the upper lid 286 is rotated and opened in a horizontal plane, and the opening of the reaction vessel 98 is exposed to the outside. Next, using the single PCR chip 80, each reaction container 98 (individually 9 mm, half of the predetermined pitch) from the corresponding one liquid storage section 64a of the PCR reagent cooling block 64 is used. Pitch in the reaction vessel 282 with the top lid by aspirating a PCR reagent, for example, the primer-containing solution, which is multiple times (4 times in this example) of the amount required for one (eg, several μl). Are accommodated by sequentially dispensing into a plurality of reaction vessels 98 of different sizes. At that time, since the amount of liquid multiple times the amount required for one reaction vessel is handled, the amount of liquid increases and it is easy to perform suction and discharge. The above steps are repeated until the necessary reagents are dispensed.

  In step S′6, after the nozzle head 12 is detached from the empty chip accommodating portion 78b of the container 78 by using the moving mechanisms 20 and 21, the single PCR chip 80 attached to the nozzle head 12 is detached. A new PCR chip 80 accommodated in the second chip accommodating portion 78a of the container 78 is attached to the end of the nozzle head 12 or the nozzle 22 arranged second, and the movement is performed. The mechanism 20 and 21 are used to move to the genome storage solution storage section 64b that stores the extracted DNA of the PCR reagent cooling block 64, and each of the DNA extracted from four patients A plurality of times (in this example, 4 times) the required amount of the DNA solution in each reaction container 98 is sucked, moved to the reaction container 98, and sequentially dispensed into the required number of containers. . The above process is repeated for all four patients.

  In step S′7, after the reaction vessel 98 with the top lid is attached to the upper surface of the temperature controller 300 with respect to the reaction vessel 98 to prevent the reaction vessel 98 from being lifted up, The pad 306 having an L-shaped cross section is inserted into the covering surface 286b of the upper lid 286 by sliding it from the side below the covering surface 286b (the pad 306 is supported on the covering surface 286b side). A support groove or the like is provided) and the rotary cylinder 287 is rotated by an opening / closing drive motor to rotate the upper lid rotating shaft 284, thereby rotating the upper lid support handle 283 and the upper lid 286. The pad 306 is rotated in a horizontal plane without contacting the opening of the reaction vessel 98 so that the opening of the reaction vessel 98 is moved upward. The cover surface 286b of the lid 86 or the pad 306 is covered.

  In step S′8, the nozzle head 12 is moved by the moving mechanisms 20 and 21 to the position of the empty chip accommodating portion 78b, and then the PCR chip 80 is removed from the nozzle 22 by the detaching mechanism ( 29, 27).

  In step S′9, as shown in FIG. 20 and FIG. 21, the rod 88 and the rod 90 are interlocked with the nozzle 22 using the suction / discharge mechanism 23 and the moving mechanism (20, 21), or In conjunction with the piston in the cylinder, after being lowered to a predetermined height with respect to the stage 214, the X and Y axis moving mechanism 20 moves along the X axis direction to the reaction vessel 282 with the upper lid 86 where the upper lid 86 is located. Then, the XY axis moving mechanism 20 is used to move along the Y axis direction.

  Here, the “predetermined height” is a height at which the grooves 92 and 94 formed at the lower ends of the rods 88 and 90 can be engaged with the hook 85 of the upper lid 286.

  In step S′10, as shown in FIG. 21, the groove 92 of the rod 88 and the groove 94 of the rod 90 are fitted into the U-shaped notches 85a of the hook 85 of the upper lid 286, so that the rod The upper lid 86 is engaged with and connected to the upper cover 86 via the hooks 85 and 90.

  In step S′11, the covered surface 286b and the pad 306 are moved to the reaction vessel by pressing the pressed surface 286a of the upper lid 286 by further moving the rod 88 and the rod 90 downward. 98 openings will be sealed.

  In step S′12, the temperature controller 300 performs temperature control according to the PCR method on the reaction vessel 98. The temperature control according to the PCR method is such that the temperature of the reaction vessel 98 is set to 94 ° C. in order to denature the input double-stranded sample DNA into single-stranded DNA, and then the single-stranded DNA and In order to perform annealing or hybridization with the primer, the temperature of the reaction vessel 98 is set to 50 ° C. to 60 ° C. Next, in order to synthesize a DNA strand complementary to the single strand, the operation of setting the temperature to 74 ° C. and incubating is performed as one cycle, and is repeated a predetermined number of times, for example, about several minutes. I do.

  In step S′13, after the temperature control is completed, the rods 88 and 90 are shaken in the vertical direction or the horizontal direction (X-axis direction) using the suction / discharge mechanism 23 or the moving mechanism (20, 21) or By repeating the movement, the opening of the reaction vessel 98 is removed from the pad 306 or the pad 306 provided on the lower side of the covering surface 286b of the upper lid 286 by removing moisture due to condensation or by shifting the position. The state of being in close contact with the covering surface 286b is easily released.

  In step S′14, the upper lid 286 is opened to collect the PCR product stored in each reaction vessel 98, and various tests using the analysis chip 77 or the like can be performed using the PCR product. Analysis will be performed.

  The above embodiment has been specifically described for better understanding of the present invention, and does not limit other embodiments. Therefore, changes can be made without changing the gist of the invention. For example, it is possible to use both an operating tool such as a rod and a nozzle as the action mechanism when the upper lid is closed. In the embodiment, only the genomic PCR process has been described, but the present invention is not limited to this example, and the present invention can also be used for temperature control of other biological compounds such as proteins.

  Further, the number of nozzles or the number of rods, the type of container, its arrangement, the reagent used, the specimen, and the like are not limited to the examples shown in the embodiments. Furthermore, the configuration of the reaction container with the upper lid is not limited to the example shown in the embodiment. In the above example, the nozzle is moved with respect to the stage, but it is also possible to move the stage with respect to the nozzle.

  In addition, the reaction container with an upper lid, the temperature controller, the upper lid support member, the dispensing tip, the reaction container, the moving mechanism, the suction / discharge mechanism, the nozzle, the operating tool, the reagent, and the liquid storage unit described in each embodiment of the present invention In addition, the number, arrangement, size, and the like of the chip accommodating portion are not limited to this example. Also, these parts can be selected appropriately and combined with each other with appropriate changes.

  The present invention mainly includes, for example, biological materials such as DNA, RNA, mRNA, rRNA, tRNA, and plasmid, fields requiring examination and analysis, for example, industrial fields, agricultural fields such as food, agricultural products, and marine products processing, and pharmaceutical fields. It relates to medical fields such as hygiene, health, disease, heredity, etc., and buried fields such as biochemistry or biology. The present invention can be used particularly for processing and analysis for handling various DNAs such as PCR and real-time PCR.

Explanation of symbols

10, 110, 210 Temperature control reaction processing device 12, 112 Nozzle head 14, 114, 214 Stage 16, 116, 216 Case 19, 119 Upper lid closing mechanism 20 XY axis moving mechanism 120 Y axis moving mechanism 21, 121 Z Axis moving mechanism 22, 122 Nozzle 23, 123 Suction / discharge mechanism 24, 124 Cylinder 43, 143 Magnetic means 75, 76, 80 Perforation chip, genome extraction chip, PCR chip 82, 182, 282 Reaction vessel 86 with top cover 186,286 Upper cover 88,90 Rod (actuator)
92, 94 Groove 98, 198 Reaction vessel 100, 200, 300 Temperature controller

Claims (10)

A suction / discharge mechanism for sucking and discharging gas, and a nozzle head having one or more nozzles to which a dispensing tip capable of sucking and discharging liquid can be detachably attached by the suction / discharge mechanism;
One or more reaction vessels having an opening into which the tip of the dispensing tip can be inserted;
A moving mechanism that is relatively movable between the nozzle and the reaction vessel;
A temperature controller capable of controlling the temperature in one or more of the reaction vessels;
An upper lid provided to be openable and closable with respect to the opening of one or more of the reaction vessels whose temperature is controlled by the temperature controller;
A temperature control reaction processing apparatus, which is driven by the suction / discharge mechanism or the moving mechanism, and has an upper lid closing action mechanism that allows the upper lid to be pressed, shaken or moved when the opening is closed by the upper lid.   The suction / discharge mechanism includes a cylinder communicating with the nozzle, a piston that slides within the cylinder, and a piston drive mechanism that drives the piston. The action mechanism when the upper lid is closed is provided in the nozzle head. The temperature-controlled reaction processing apparatus according to claim 1, further comprising one or more operating tools interlocking with the piston.   3. The temperature according to claim 1, wherein the upper lid closing operation mechanism includes one or more operating tools provided in the nozzle head and interlocking with the nozzle driven by the moving mechanism. Control reaction processing equipment.   The temperature control reaction processing apparatus according to claim 1, wherein the upper lid closing mechanism has one or more nozzles driven by the moving mechanism. 5. The hook according to claim 2, further comprising a hook engageable with the operation tool or the nozzle of the upper lid closing mechanism that is driven by the moving mechanism or the suction / discharge mechanism. A temperature-controlled reaction processing apparatus according to claim 1. A shaft support portion attached to the stage provided with the reaction vessel; and an upper lid support member that is pivotally supported by the shaft support portion and elastically supports the upper lid.
The upper lid has a pressed surface that is pressed by the action mechanism when the upper lid is closed, a covering surface that is formed so as to face the pressed surface, and covers the opening of the reaction vessel. The temperature control reaction processing apparatus according to any one of claims 1 to 5, wherein the upper lid is supported in a state of being elastically biased along a direction from the covering surface toward the pressed surface. By a moving mechanism that can relatively move between one or two or more nozzles provided in the nozzle head and one or two or more reaction vessels, and a suction / discharge mechanism that performs gas suction / discharge provided in the nozzle head, 1 or 2 or more in which the temperature of the liquid sucked into the dispensing tip removably attached to one or more of the nozzles can be controlled, and the upper lid that can be opened and closed with respect to the opening is opened. An accommodating step of discharging and accommodating in the reaction container;
Closes the opening portion of one or more of said reaction vessel by said upper lid, the lid closed when the action mechanism driven by said suction and discharge mechanism or the moving mechanism, the reaction vessel by pressing the upper lid from above A clogging step for temperature control;
An opening step of opening the opening of the reaction container by shaking or moving the upper cover closed with respect to the opening of the reaction container by the action mechanism when the upper cover is closed driven by the suction / discharge mechanism or the moving mechanism And a temperature-controlled reaction processing method. The closing step, the nozzle where the suction upper lid closed when the action mechanism driven by a discharge mechanism with one or more of the working tool having the or wherein Ru is driven by a moving mechanism lid closed when the action mechanism has Or, using one or more actuators provided in the nozzle head and interlocking with the nozzle, pressing the upper lid closing the opening from above,
The temperature control reaction processing method according to claim 7, wherein in the opening step, the upper lid that closes the opening is shaken or moved using the nozzle or the operating tool. A desorption step of desorbing the dispensing tip from the nozzle, the desorption step,
The temperature control reaction processing method according to claim 7, wherein the temperature control reaction processing method is executed in the housing step or after the housing step and before the closing step.   The said accommodating process is performed by mounting | wearing with one said dispensing tip only to one said nozzle among the several said nozzles arranged in the said nozzle head. Temperature control reaction processing method.

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