JP6098186B2 - Reactor - Google Patents

Description

  The present invention relates to a reaction apparatus used for reacting a test piece on which a physiologically active substance such as a nucleic acid is immobilized with a specimen.

  In recent years, with the progress of genome sequencing, all the base sequences of the genome of each organism have been clarified. An inspection method using a nucleic acid microarray is known as a technique for analyzing the type of a gene using the revealed base sequence. For example, it is expected to contribute to diagnosis and treatment by analyzing fungal genes and specifying the type of drug resistance.

  Since the nucleic acid microarray only needs to be able to qualitatively detect a specific base sequence, an inexpensive nucleic acid microarray based on a piece of paper such as nitrocellulose or nylon is desired (Patent Document 1). In order to simplify the work of reacting a nucleic acid microarray with a substance to be inspected, there has been proposed an apparatus that causes a machine to perform dispensing and washing of reagents (Patent Document 2).

JP 7-503143 A JP 2003-57245 A

  However, the processing apparatus disclosed in Patent Document 2 has a problem of lack of versatility because a nucleic acid microarray having a shape and the like is necessary. In addition, there is a problem that the structure of the apparatus is complicated and it is difficult to reduce the cost, for example, it is necessary to move the nozzle of the reagent in two directions and to provide a flow path for waste liquid in each processing tank.

  This invention is made | formed in view of such a situation, The objective is to provide the reaction apparatus of versatility and a simple structure.

  (1) In the reaction apparatus according to the present invention, a slender reaction tank capable of storing a test sample having a strip shape and storing a liquid having a capacity for immersing the test piece has a first direction along the longitudinal direction of the reaction tank. A plurality of reaction units arranged in parallel in a second direction orthogonal to the above, and a reagent unit having a reagent nozzle that is movable relative to the reaction tank in the second direction and dispenses the reagent to the reaction tank And a cleaning part that is movable relative to the reaction tank in the second direction and has a cleaning nozzle for dispensing a cleaning liquid into the reaction tank, and relative to the reaction tank in the second direction. And a waste liquid part having a waste liquid nozzle for sucking the liquid stored in the reaction tank.

  For example, a test piece, which is a nucleic acid microarray, is put into each reaction tank and accommodated. A test substance and a reagent are injected into each reaction tank. The test substance is, for example, DNA amplified by PCR, and is injected into each reaction tank by the user. The reagent is a reaction liquid for hybridizing the test substance and the nucleic acid microarray, and is dispensed from the reagent part to each reaction tank through the reagent nozzle. After the time for hybridization has elapsed, the reagent is aspirated and discharged from each reaction tank by the waste liquid section through the waste liquid nozzle. Thereafter, the cleaning liquid is dispensed from the cleaning section to each reaction tank through the cleaning nozzle. After the test piece is washed with the washing solution, in the same manner, for example, a conjugate solution labeled with an enzyme is dispensed into each reaction vessel, reacted, washed again, and then the substrate solution that develops color with the enzyme. Is dispensed into each reaction vessel and reacted. Since the finally cleaned test strip remains in each reaction tank, the type of the test substance is identified by visually observing the color development state of the test strip.

  (2) The reaction unit may include a tray provided with the plurality of reaction vessels and a base on which the tray is placed.

  Since the tray can be attached to and detached from the base, the tray can be made disposable.

  (3) The base may change its posture so that the inclination angle along the first direction changes, and may further include a drive source for changing the posture of the base.

  Since each reaction tank of the tray also tilts due to the inclination of the base, the reagent or the cleaning liquid is stirred in each reaction tank in which the test piece is accommodated.

  (4) The base includes a heat source, and may further include a control unit that controls the heat generation temperature of the heat source.

  For example, in the reaction between the test substance and the test piece, each reaction tank can be maintained at a temperature suitable for hybridization.

  (5) The reaction vessel is provided at a position close to one end side in the first direction, and has a protruding piece protruding so as to narrow the width of the reaction vessel along the second direction. The waste liquid nozzle may suck the liquid stored in the reaction tank on one end side in the first direction from the protruding piece.

  In each reaction tank, there is a protruding piece between the space where the test piece is accommodated and the space where the waste liquid nozzle enters, so that the test piece moves to the waste liquid nozzle as the reagent or cleaning liquid is sucked. The clogging of the test piece in the waste liquid nozzle is prevented.

  According to the reaction apparatus according to the present invention, since the plurality of elongated reaction vessels are arranged in parallel in the direction orthogonal to the longitudinal direction, the reaction unit can be reduced in size. In addition, since the reagent nozzle, the washing nozzle, and the waste liquid nozzle move relatively in a certain direction with respect to each reaction tank that accommodates each test piece, the structure of the apparatus is simplified. As a result, a reactor having versatility and a simple structure is realized.

FIG. 1 is a perspective view showing an external configuration of a reaction apparatus 10 according to the embodiment. FIG. 2 is a central longitudinal sectional view of the reaction unit 20 along the left-right direction. FIG. 3 is a plan view of the tray 22. 4 is a cross-sectional view taken along the line IV-IV in FIG. FIG. 5 is an enlarged plan view of the reaction vessel 25.

  Hereinafter, preferred embodiments of the present invention will be described. In addition, this embodiment is only one embodiment of this invention, and it cannot be overemphasized that an embodiment can be changed in the range which does not change the summary of this invention.

[Structure of reactor 10]
As shown in FIG. 1, the reaction apparatus 10 has a substantially rectangular parallelepiped outer shape that can be installed on a table. The reactor 10 can be accessed normally from the front side and the top side of the device. A part of the front side and the upper side of the reaction apparatus 10 can be opened and closed by a cover 11 that is rotatably attached to the upper wall of the apparatus housing. As shown in FIG. 1, the inside of the reaction apparatus 10 can be accessed by turning the cover 11 upward to the open position. Although not shown in the drawings, the reaction unit 20 and the like are covered with the cover 11 when the cover 11 is in the closed posture. Accordingly, there are advantages that dust can be prevented from entering the reaction unit 20 or the like when the reaction apparatus 10 is operating, or the reaction unit 20 can be kept warm.

  An operation panel 12 is provided on the right side of the front wall of the apparatus housing of the reaction apparatus 10. The operation panel 12 includes a display and buttons. Through the operation panel 12, the user can make a desired input to the reaction apparatus 10, and can confirm the input result, the operation state, and the like. Although not shown in each figure, the operation panel 12 is electrically connected to a control board provided inside the reaction apparatus 10. The control board constitutes an arithmetic unit and includes a CPU, a ROM, a RAM, and the like. A control unit is configured by the control board.

  The lower part of the front wall of the apparatus housing of the reactor 10 is opened. A plurality of bottles filled with a reagent and a cleaning liquid are placed in the internal space 13 continuous with the opening. Although not shown in each figure, one end of a plurality of tubes that are continuous with each nozzle of the dispensing waste liquid unit 30 is projected into the internal space 13. By inserting each tube into each bottle placed in the internal space 13, the reagent and the cleaning liquid are supplied from each bottle to each nozzle of the dispensing waste liquid unit 30.

  As shown in FIG. 1, a reaction unit 20 is provided inside the reaction apparatus 10. As shown in FIGS. 1 and 2, the reaction unit 20 includes a base 21 and a tray 22. The base 21 has a flat plate shape that is elongated in the left-right direction. The tray 22 is installed on the upper side of the base 21 so as to cover the upper surface of the base 21. A heat source 23 is provided inside the base 21. The heat source 23 is connected to the control board described above, and generates heat when electric power is supplied from the control board at a predetermined timing. Heat generated by the heat source 23 is transmitted to the tray 22 through the base 21. The reaction unit 20 corresponds to the reaction unit.

  On the rear side of the base 21, shafts 24 that protrude in the left-right direction are provided. A shaft 24 is rotatably fixed to the apparatus housing. Although not shown in each figure, the base 21 is centered on the shaft 24 by being transmitted to the shaft 24 from a drive source provided inside the apparatus and controlled by a control board. The pendulum moves with the front side opposite to the shaft 24 in the front-rear direction as a tip. By this pendulum movement, the base 21 changes its posture so that the upper surface on which the tray 22 is placed changes the inclination angle with respect to the horizontal direction. Thereby, the liquid in the reaction tank 25 of the tray 22 placed on the base 21 is agitated.

  As shown in FIGS. 3 and 4, the tray 22 has a flat plate shape that is elongated in the left-right direction. The edge 26 of the tray 22 is slightly larger than the edge of the upper surface of the base 21. The tray 22 is positioned on the upper surface side of the base 21 so that the edge 26 fits outside the base 21. The tray 22 is a molded product made of resin, and a reaction tank 25 that is recessed downward from the upper surface 27 is formed.

  The reaction vessel 25 has an elongated groove shape that accommodates a strip-shaped test piece and can store a liquid having a capacity for immersing the test piece. In the state where the tray 22 is placed on the base 21, the longitudinal direction of the reaction tank 25 is along the front-rear direction. A plurality of the reaction tanks 25 are formed in parallel in the left-right direction. The plurality of reaction tanks 25 have the same shape except for the arrangement. The front-rear direction corresponds to the first direction, and the left-right direction corresponds to the second direction.

  In the reaction tank 25, both ends in the longitudinal direction (front-rear direction) are close to the edge 26 of the tray 22. In the state where the tray 22 is mounted on the base 21 among the both ends in the longitudinal direction, it protrudes so as to narrow the width of the reaction tank 25 along the left-right direction close to one end side which is the rear side in the front-rear direction. The protruding piece 28 is formed. The protruding piece 28 protrudes from one side wall (the right side wall in FIG. 3) in the left-right direction in the reaction tank 25 toward the other wall (the left side wall in FIG. 3), and the protruding end thereof protrudes from the other wall. It is close. The width of the portion narrowed by the projecting piece 28 is narrower than the width of the strip-shaped test piece. Therefore, the test piece accommodated on the other end side which is the front side of the reaction tank 25 does not move to the rear end by the projecting piece 28 in a posture in which the width direction is substantially along the horizontal direction. On the other hand, since the projecting piece 28 does not completely divide the reaction tank 25, liquid can flow through the gap between the projecting end of the projecting piece 28 and the wall of the reaction tank 25.

  As shown in FIG. 1, a dispensing waste liquid unit 30 is provided on the upper side of the tray 22. The dispensing waste liquid unit 30 has a front end located immediately above the tray 22 and a rear end extending rearward from the tray 22. The rear end side of the dispensing waste liquid unit 30 is supported by the guide rail 37, and is connected to the belt drive mechanism 38. Although not shown in detail in each figure, the belt drive mechanism 38 has a drive pulley and a driven pulley that are rotated by a stepping motor and are arranged in the left-right direction, and is arranged between the drive pulley and the driven pulley. An endless belt is laid. The dispensing waste liquid unit 30 is connected to the endless belt of the belt drive mechanism 38, and the dispensing waste liquid unit 30 moves in the left-right direction by rotating the drive pulley. The driving of the belt driving mechanism 38 is controlled by the control board.

  The dispensing waste liquid unit 30 includes a first reagent nozzle 31, a second reagent nozzle 32, a third reagent nozzle 33, a cleaning nozzle 34, a purified water nozzle 35, and a waste liquid nozzle 36. As shown in FIG. 4, each nozzle is disposed immediately above one reaction tank 25 of the tray 22. Among the nozzles, the first reagent nozzle 31, the second reagent nozzle 32, the third reagent nozzle 33, the washing nozzle 34, and the purified water nozzle 35 are disposed in front of the projecting piece 28 of the reaction tank 25, and are disposed of waste liquid. The nozzle 36 is disposed behind the projecting piece 28 of the reaction tank 25. Only the waste liquid nozzle 36 is configured to be movable in the vertical direction with respect to the tray 22. In the posture in which the waste liquid nozzle 36 is moved upward, the tip of the waste liquid nozzle 36 is located above the upper surface 27 of the tray 22 (as shown in FIG. 4), and in the posture in which the waste liquid nozzle 36 is moved downward. The tip of the waste liquid nozzle 36 is located near the bottom of the reaction tank 25.

  Each nozzle is not necessarily arranged directly above one reaction tank 25. For example, a group of the first reagent nozzle 31, the second reagent nozzle 32, and the third reagent nozzle 33, the washing nozzle 34, The purified water nozzle 35 and the group of waste liquid nozzles 36 may be separately arranged immediately above the two adjacent reaction tanks 25. Further, for example, a plurality of waste liquid nozzles 36 may be provided so as to be positioned immediately above the plurality of reaction vessels 25.

  Moreover, although not appearing in each figure, each nozzle is connected to each bottle or waste liquid tank placed in the internal space 13 via a tube. Each tube is provided with a tube pump, and has a structure in which liquid can be dispensed and sucked from each nozzle by driving the tube pump. A group of these pumps, tubes, and nozzles constitutes a reagent part, a washing part, and a waste liquid part.

[Operation of reactor 10]
The reaction apparatus 10 is used for a qualitative test using, for example, a test piece 40 (see FIG. 5) that is a nucleic acid microarray. The nucleic acid microarray is used for identifying, for example, Mycobacterium tuberculosis and HCV. A qualitative test using a nucleic acid microarray is disclosed in, for example, Japanese Patent Application Laid-Open No. 2011-177186. Hereinafter, the operation of the reaction apparatus 10 will be described by taking a qualitative test using a nucleic acid microarray (test piece 40) for identifying Mycobacterium tuberculosis as an example. A plurality of types of DNA probes capable of hybridizing with a specific base sequence of Mycobacterium tuberculosis are fixed to the test piece 40. As the test piece 40, an organic material such as nitrocellulose is typically used. The test piece 40 has an elongated strip shape and is a size that can be accommodated in the reaction vessel 25.

  Prior to the qualitative test using the test piece 40, DNA as a test substance is prepared. DNA is extracted from a human body fluid, a culture strain, etc. by a well-known method, for example. The extracted DNA is amplified by a known technique such as PCR. The ends of the primers used for PCR are modified with, for example, biotin.

  Prior to the qualitative test using the test piece 40, reaction conditions are input to the operation panel 12 of the reaction apparatus 10. The reaction conditions include hybridization, enzyme modification, time and temperature in the substrate reaction, the amount of each reagent injected, the stirring time of the tray 22 and the number of washings.

  First, a new tray 22 that has not been subjected to a reaction is placed on the base 21. Although a detailed description is omitted, a tray 22 is fixed to the base 21 using a fixture. The test piece 40 is put into the reaction tank 25 of the tray 22. The test pieces 40 are put into the plurality of reaction tanks 25 by the number of test substances, respectively. Further, DNA as a test substance is also injected into each reaction tank 25.

  When the operation of the reaction apparatus 10 is started, the operation is automatically performed in the order of hybridization, washing, enzyme modification, washing, substrate reaction, washing, and drying. In the hybridization, the heat source 23 of the base 21 is heated to 62 ° C. Then, the hybridizing solution is dispensed from the first reagent nozzle 31 to the reaction tank 25. When there are a plurality of test pieces 40, the belt driving mechanism 38 is driven, the first reagent nozzle 31 is moved to the position corresponding to each reaction tank 25 in the left-right direction, and the hybridizing solution is dispensed to each reaction tank 25. The Then, while the tray 22 is shaken by the pendulum movement of the base 21, the reaction is performed by being left for 30 minutes, for example, for the time necessary for hybridization.

  After the time necessary for hybridization has elapsed, the heat source 23 is stopped and returned to room temperature. Moreover, the test piece 40 in each reaction tank 25 is washed. In the cleaning, first, the waste nozzle 36 is moved downward toward the predetermined reaction tank 25. Then, the hybridization liquid (including DNA as the test substance) stored in the reaction tank 25 is aspirated. Since the tip of the waste liquid nozzle 36 is located behind the projecting piece 28 in the reaction tank 25 and the test piece 40 is located in front of the projecting piece 28 in the reaction tank 25, it is separated by the projecting piece 28. When the hybridizing liquid is sucked from the reaction tank 25, the flow of the hybridizing liquid suppresses the movement of the test piece 40 to the vicinity of the tip of the waste liquid nozzle 36, and only the hybridizing liquid is discharged from the reaction tank 25. The

  After the hybridizing liquid is discharged from the reaction tank 25, the waste liquid nozzle 36 is moved upward, and the cleaning liquid is dispensed from the cleaning nozzle 34 to the reaction tank 25. Then, after the tray 22 is shaken only for a predetermined time, for example, for 1 minute, the waste liquid nozzle 36 is moved downward, and the cleaning liquid is sucked and discharged from the reaction tank 25. Such washing is repeated several times. When there are a plurality of test pieces 40, the belt driving mechanism 38 is driven, and the waste liquid nozzle 36 and the washing nozzle 34 are moved to the positions corresponding to the reaction tanks 25 in the left-right direction, and the same washing is performed. The cleaning solution is a buffer solution containing a surfactant, for example.

  After the test piece 40 is washed with the washing liquid, the conjugate liquid is dispensed from the second reagent nozzle 32 to the reaction tank 25. When there are a plurality of test pieces 40, the belt drive mechanism 38 is driven, the second reagent nozzle 32 is moved to the position corresponding to each reaction tank 25 in the left-right direction, and the conjugate liquid is dispensed to each reaction tank 25. The Then, while the tray 22 is shaken by the pendulum movement of the base 21, the reaction is performed by leaving it for 30 minutes, for example, for the time necessary for the biotin-modified primer and the conjugate to react. The conjugate is, for example, streptavidin-modified alkaline phosphatase, and the biotin-modified primer reacts with the conjugate through an avidin-biotin bond.

  After the time necessary for the reaction of the conjugate elapses, the test piece 40 in each reaction tank 25 is washed. Cleaning is performed by the same operation as described above. After washing, purified water is injected from the purified water nozzle 35 to each reaction tank 25 and shaken for several minutes, and then the purified water is discharged from the reaction tank 25 by the waste liquid nozzle 36.

  After the test piece 40 is washed with purified water, the substrate solution is dispensed from the third reagent nozzle 33 to the reaction tank 25. When there are a plurality of test pieces 40, the belt drive mechanism 38 is driven, the third reagent nozzle 33 is moved in the left-right direction to a position corresponding to each reaction tank 25, and the substrate solution is dispensed to each reaction tank 25. . Then, while the tray 22 is shaken by the pendulum movement of the base 21, the reaction is performed by being left for 30 minutes, for example, for the time required for the enzyme reaction. The substrate solution is, for example, p-nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt.

  After the time required for the enzyme reaction has elapsed, the test piece 40 in each reaction tank 25 is washed with purified water. Washing with purified water is performed by the same operation as described above. After cleaning, the heat source 23 of the base 21 is heated and the test piece 40 in the reaction tank 25 is dried.

  When the drying is finished, the operation of the reactor 10 is stopped. The user removes the tray 22 from the base 21, takes out the test piece 40 accommodated in each reaction tank 25, and visually confirms the color development state. If the DNA as the test substance has base sequences corresponding to a plurality of types of DNA probes fixed to the test piece 40, the positions corresponding to the DNA probes are colored. For example, if Mycobacterium tuberculosis present in the test substance is mutated, the base sequence corresponding to the DNA probe is mutated and the position corresponding to the DNA probe does not develop color. By confirming the color development state of the test piece 40 as described above, it is possible to qualitatively determine the mutation of the test substance DNA.

[Operational effects of this embodiment]
According to the reaction apparatus 10 according to the present embodiment, the plurality of elongated reaction tanks 25 are arranged in parallel in the direction (left-right direction) perpendicular to the longitudinal direction (front-rear direction), so that the reaction unit 20 can be downsized. Realized. Moreover, since each nozzle moves to a fixed direction (left-right direction) with respect to each reaction tank 25 which accommodates each test piece 40, the structure of an apparatus is simplified. Thereby, the reaction apparatus 10 having versatility and a simple structure is realized.

  Further, the reaction unit 20 includes a tray 22 provided with a plurality of reaction vessels 25 and a base 21 on which the tray 22 is placed, and the tray 22 can be attached to and detached from the base 21. The tray 22 can be made disposable.

  Further, the posture of the base 21 can be changed so that the tilt angle changes with respect to the horizontal direction, and each reaction tank 25 of the tray 22 is also tilted by the tilt of the base 21, so that each test piece 40 accommodated is accommodated. In the reaction tank 25, the reagent or the cleaning liquid is stirred.

  Moreover, since the base 21 has the heat source 23, each reaction tank 25 can be kept at a temperature suitable for hybridization in the reaction between the test substance and the test piece 40.

The reaction tank 25 is provided at a position close to the rear side in the front-rear direction, and has a protruding piece 28 protruding so as to narrow the width of the reaction tank 25 along the left-right direction. 36 sucks the liquid stored in the reaction tank 25 on the rear side in the front-rear direction from the projecting piece 28.
In each reaction tank 25, a projecting piece 28 exists between a space in which the test piece 40 is accommodated and a space into which the waste liquid nozzle 36 enters. As a result, the test piece 40 is moved to the tip of the waste liquid nozzle 36 in the reaction tank 25 as the reagent or the cleaning liquid is sucked, and the waste liquid nozzle 36 is prevented from being clogged.

10 ... Reaction device 20 ... Reaction unit (reaction unit)
21 ... Base 22 ... Tray 23 ... Heat source 25 ... Reaction tank 28 ... Projection piece 31, 32, 33 ... Reagent nozzle 34 ... Cleaning nozzle 36 ... Waste liquid nozzle 40 ... Test piece

Claims (4)

A plurality of elongate reaction tanks that can store a liquid having a capacity for storing strip-shaped test pieces and immersing the test pieces are juxtaposed in a second direction perpendicular to the first direction along the longitudinal direction of the reaction tanks. Reaction part,
A reagent part movable relative to the reaction tank in the second direction and having a reagent nozzle for dispensing a reagent into the reaction tank;
A washing part having a washing nozzle that is movable relative to the reaction tank in the second direction and dispenses a washing liquid into the reaction tank;
A waste liquid part that is movable relative to the reaction tank in the second direction and has a waste liquid nozzle for sucking the liquid stored in the reaction tank ;
The reaction tank is provided at a position close to one end side in the first direction, and has a protruding piece protruding so as to narrow the width of the reaction tank along the second direction,
The said waste liquid nozzle is a reaction apparatus which attracts | sucks the liquid stored in the said reaction tank in the one end side of the said 1st direction from the said protrusion .   The reaction apparatus according to claim 1, wherein the reaction unit includes a tray provided with the plurality of reaction vessels and a base on which the tray is placed.   The reaction apparatus according to claim 2, wherein the base is capable of changing its posture such that an inclination angle along the first direction changes, and further includes a drive source for changing the posture of the base.   The reaction apparatus according to claim 2 or 3, wherein the base includes a heat source, and further includes a control unit that controls an exothermic temperature of the heat source.

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