JP4540996B2 - Nucleic acid detector - Google Patents

Description

  The present invention relates to a nucleic acid detection apparatus that detects nucleic acids.

  Conventionally, nucleic acid detection apparatuses that detect nucleic acids are known (see, for example, Patent Document 1 and Patent Document 2). In the above-mentioned Patent Document 1, a part of the amplified sample is aspirated from the inside of the disposable container with the lid closed, and incubated in the well without the lid with the top open, and the detection cell without the lid with the top opened is used. A technique for dispensing a sample to detect a nucleic acid is disclosed.

Patent Document 2 discloses a device that amplifies and detects nucleic acid while opening and closing a lid provided separately from the reaction vessel with the lid removing device.
WO95 / 11083 publication Japanese Patent No. 3051649

  However, in the technique disclosed in Patent Document 1, since the lid is not provided for the well in which incubation is performed and the detection cell in which detection is performed, the sample scatters during the incubation or detection process, so that other samples or There is a problem that the reagent may be contaminated.

  Further, in the apparatus disclosed in Patent Document 2, since the reaction container and the lid are separate, when the lid of the reaction container is removed and lifted by the lid removing device, the lid can drop onto another container. There is sex. In this case, there is a problem that the sample and the reagent in the other container are contaminated by the contaminated lid. In addition, even when the lid does not fall, there is a problem that the sample or reagent in the other container may be contaminated by the liquid droplets adhering to the lid lifted by the removing device falling into the other container. There is a point.

  The present invention has been made to solve the above-described problems, and an object of the present invention is to provide a nucleic acid detection apparatus capable of preventing a sample or a reagent from being contaminated.

Means for Solving the Problems and Effects of the Invention

In order to achieve the above object, a nucleic acid detection device according to a first aspect of the present invention includes a detection container mounting portion provided with a mounting hole for mounting a detection container provided with a lid integrally, and a detection A lid closing member for placing the lid of the detection container placed in the placement hole and pivotable with respect to the container placement portion, and a reagent on the detection container placed in the placement hole And a dispensing means for dispensing a sample suspected of containing the target nucleic acid, and a lid closing member so as to close the opening of the detection container with a lid after dispensing of the reagent and sample into the detection container is completed. A drive unit for driving , an amplifying unit for amplifying the target nucleic acid in the detection container whose lid is closed by the lid closing member, and a detection unit for detecting the presence of the target nucleic acid in the detection container whose lid is closed Yes.

  In the nucleic acid detection apparatus according to the first aspect, as described above, a lid closing means for closing the lid of the detection container after the dispensing of the reagent and the sample into the detection container is provided, and the lid of the detection container is closed. In this state, by providing an amplification means for amplifying the target nucleic acid and a detection means for detecting the presence of the target nucleic acid, the lid of the detection container is closed at the time of amplification and detection. Thus, contamination of other samples and reagents can be prevented. In addition, by using a detection container provided with an integrated lid, the lid does not fall on other detection containers, so that samples in other detection containers are contaminated due to the fall of the lid. There is no inconvenience.

  In the nucleic acid detection apparatus according to the first aspect described above, preferably, the dispensing unit is connected to the nozzle unit to which the dispensing chip is detachably attached to the tip thereof, and sucks and discharges the sample and the reagent. And a pump part for. If comprised in this way, since a dispensing tip can be replaced | exchanged for every sample, contamination (contamination) can be prevented.

  In the nucleic acid detection device according to the first aspect described above, preferably, the detection means includes a light source that irradiates light to the liquid in the detection container, and a light detection unit that detects light emitted from the light source. If comprised in this way, presence of the target nucleic acid in a detection container can be easily detected with the detection means which consists of a light source and a photon detection part.

  In the nucleic acid detection apparatus according to the first aspect, preferably, the detection means detects the presence of the target nucleic acid by detecting the turbidity of the mixed solution of the sample and the reagent in the detection container. If comprised in this way, presence of a target nucleic acid can be detected easily.

  In this case, it is preferable that the detection means detects the presence of the target nucleic acid by detecting a change in turbidity of the mixed liquid of the sample and the reagent in the detection container. If comprised in this way, presence of a target nucleic acid can be detected easily based on the change of the detected turbidity.

  In the nucleic acid detection apparatus according to the first aspect, preferably, the detection means detects the presence of the target nucleic acid by detecting a reagent that binds to the amplification product of the target nucleic acid. If comprised in this way, presence of a target nucleic acid can be detected easily.

  In the nucleic acid detection apparatus according to the first aspect, preferably, the amplification of the target nucleic acid by the amplification means and the detection of the target nucleic acid by the detection means are performed in parallel. If comprised in this way, the time of amplification and detection processing of a target nucleic acid can be shortened.

  In the nucleic acid detection apparatus according to the first aspect, the target nucleic acid may be a cytokeratin nucleic acid.

  In the nucleic acid detection apparatus according to the first aspect, the amplification of the target nucleic acid by the amplification means is preferably performed by the LAMP method. If the LAMP method is used in this way, the target nucleic acid can be efficiently amplified, so that the time required for detection of the target nucleic acid can be shortened.

In the nucleic acid detection device according to the first aspect, preferably, the dispensing of the reagent and the sample into the detection container by the dispensing means is performed with the lid of the detection container being opened. If comprised in this way, the reagent and sample can be easily dispensed to the detection container by the dispensing means.

In the nucleic acid detection device according to the first aspect, preferably, shut the lid by a lid closing member is performed only once for a single detection cell. If comprised in this way, since there is no process of opening a lid | cover after lid | cover closing operation | movement, a process can be speeded up that much.

In the nucleic acid detection device according to the first aspect described above, preferably, the lid closing member is operated to press the lid closing member downward from the upper side, thereby applying a pressing force from above to the lid, and performing the lid closing operation of the lid by the lid closing member. A pressing member to be completed is further included. If comprised in this way, the lid | cover closing operation | movement of a detection container can be reliably performed by the lid | closing operation | movement by the pressing force from the upper direction by a pressing member.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

(First embodiment)
FIG. 1 is a perspective view showing the overall configuration of the nucleic acid detection device and its peripheral devices according to the first embodiment of the present invention. 2 is a perspective view showing the overall configuration of the measurement unit of the nucleic acid detection device shown in FIG. 1, and FIG. 3 is a schematic plan view of FIG. 4 to 15 are diagrams showing details of the components of the measurement unit of the nucleic acid detection device shown in FIG. In the first embodiment, a gene amplification detection apparatus will be described as an example of the nucleic acid detection apparatus of the present invention. The gene amplification detection apparatus according to the first embodiment is an analysis apparatus that supports diagnosis of cancer metastasis in a resected tissue in cancer surgery, and a cancer-derived gene (mRNA) present in the resected tissue is LAMP (Loop-mediated Isothermal Amplification). , Eiken Chemical Co., Ltd.), and the detection is performed by measuring the turbidity of the solution generated by the amplification. Details of the LAMP method are disclosed in US Pat. No. 6,410,278.

  First, the overall configuration of the nucleic acid detection device (gene amplification detection device) of the first embodiment and its peripheral devices will be described with reference to FIG. As shown in FIG. 1, a nucleic acid detection apparatus (gene amplification detection apparatus) 100 according to the first embodiment includes a measurement unit 101 and a data processing unit 102 connected to the measurement unit 101 via a communication line. Yes. The data processing unit 102 includes a personal computer including a keyboard 102a and a mouse 102b. In addition, a printer 200 as a peripheral device and a host computer 300 are connected to the data processing unit 102 via a communication line. The printer 200 is provided for printing graphic data and text data. In addition, measurement data is output from the data processing unit 102 to the host computer 300.

  As shown in FIGS. 2 and 3, the measuring unit 101 includes a dispensing mechanism unit 10, a sample container setting unit 20, a reagent container setting unit 30, a chip setting unit 40, a chip discarding unit 50, and five And a reaction detection unit 60 including a reaction detection block 60a. The dispensing mechanism unit 10 is an example of the “dispensing unit” in the present invention, and the reaction detection unit 60 is an example of the “amplifying unit” and the “detecting unit” in the present invention. In addition, as shown in FIG. 2, the measurement unit 101 controls the apparatus by a microcomputer and supplies power to the entire apparatus including the control unit 70 that controls input / output with the outside of the apparatus and the control unit 70. A power supply unit 80 is incorporated. An emergency stop switch 90 is installed at a predetermined location in front of the measurement unit 101.

  The dispensing mechanism unit 10 has an arm unit 11 that can move in the X-axis and Y-axis directions (plane direction), and 2 that can move in the Z-axis direction (vertical direction) independently of the arm unit 11. A series (two) of syringe sections 12 are included. As shown in FIG. 4, the syringe part 12 includes a nozzle part 12a to which a pipette tip 41 (to be described later) is attached at the tip, a pump part 12b for performing suction and discharge, and a motor 12c serving as a drive source for the pump part 12b. The liquid level detection sensor 12d and the pressure detection sensor 12e are included. In the pump unit 12b, the suction and discharge functions are obtained by converting the rotation of the motor 12c into a piston motion. The liquid level detection sensor 12d is a capacitance type sensor, and detects that the tip of the pipette tip 41 made of a conductive resin is in contact with the liquid level. Moreover, the pressure detection sensor 12e detects the pressure at the time of attraction | suction and discharge by the pump part 12b. The liquid level detection sensor 12d and the pressure detection sensor 12e detect whether suction and discharge are reliably performed.

  As shown in FIGS. 2 and 3, a sample container setting base 21 having five sample container setting holes 21 a and a gripping part 21 b can be removed in a recess (not shown) of the sample container setting part 20. It is inserted in. The five sample container setting holes 21a of the sample container setting table 21 are provided at predetermined intervals in one row along the X-axis direction. A sample container 22 containing a solubilized extract (sample) prepared by processing (homogenizing, filtering, diluting) the excised tissue in advance is set in the five sample container setting holes 21a of the sample container setting table 21. Is done. In the sample container setting hole 21a, a container containing a calibrator containing a target gene having a predetermined concentration serving as a reference for creating a calibration curve, which will be described later, or a gene that should not be amplified is not normally amplified. A container containing a negative control for confirmation is also arranged.

  In addition, a reagent container setting base 31 having two primer reagent container setting holes 31a, two enzyme reagent container setting holes 31b, and a gripping part 31c is removed from a recess (not shown) of the reagent container setting part 30. It has been fitted. Two primer reagent container setting holes 31a and two enzyme reagent container setting holes 31b of the reagent container setting table 31 are provided at predetermined intervals along the Y-axis direction. The primer reagent container setting hole 31a and the enzyme reagent container setting hole 31b of the reagent container setting table 31 store primer reagent containers 32a containing two types of primer reagents and enzyme reagents corresponding to the two types of primer reagents. Each of the two enzyme reagent containers 32b is set. In the first embodiment, the primer reagent container 32a containing the primer reagent of cytokeratin 19 (CK19) and the enzyme reagent of CK19 are accommodated in the primer reagent container set hole 31a and the enzyme reagent container set hole 31b on the left side of the front. The enzyme reagent container 32b thus arranged is arranged. Also, a primer reagent container 32a in which a primer reagent for β-actin is accommodated in a primer reagent container set hole 31a and an enzyme reagent container set hole 31b on the right side of the front, and an enzyme in which an enzyme reagent for β-actin is accommodated. A reagent container 32b is arranged.

  In addition, two racks 42 each having a storage hole 42a capable of storing 36 pipette tips 41 are detachably fitted in two recesses (not shown) of the chip set unit 40, respectively. In addition, the chip set unit 40 is provided with two removal buttons 43. By pushing the removal button 43, the rack 42 becomes removable. As shown in FIG. 5, the pipette tip 41 is made of a carbon-containing conductive resin material, and a filter 41a is mounted therein. The filter 41a has a function of preventing erroneous inflow of the liquid into the syringe unit 12. The pipette tip 41 is irradiated with an electron beam in a boxed state before shipment so that a degrading enzyme such as human saliva that may adhere in the manufacturing process of the pipette tip 41 does not adversely affect gene amplification. It has been subjected. The pipette tip 41 is an example of the “dispensing tip” in the present invention. Further, the rack 42 in which the pipette tips 41 are stored is stored in a state in which the lower cover 44 and the upper cover 45 are attached as shown in FIG.

  As shown in FIG. 3, the tip discarding section 50 is provided with two tip discarding holes 50 a for discarding the used pipette tips 41. Further, a groove 50b having a width narrower than that of the chip disposal hole 50a is provided so as to be continuous with the chip disposal hole 50a.

  Here, in the first embodiment, each reaction detection block 60a of the reaction detection unit 60 includes a reaction unit 61, two turbidity detection units 62, and a lid closing mechanism unit 63 as shown in FIG. Has been. The reaction unit 61 is an example of the “amplifying unit” in the present invention, and the turbidity detection unit 62 is an example of the “detecting unit” in the present invention. As shown in FIG. 3, each reaction unit 61 is provided with two detection cell setting holes 61 a for setting the detection cell 65. The detection cell 65 is an example of the “detection container” in the present invention. Further, as shown in FIG. 7, each reaction portion 61 is provided with a light irradiation groove 61b so as to expose the detection cell set hole 61a. Further, a Peltier module 61c and a heat dissipation heat sink 61d for controlling the liquid temperature inside the detection cell 65 to about 20 ° C. to about 65 ° C. are provided.

  Further, in the first embodiment, the turbidity detection unit 62 is an LED composed of a blue LED having a wavelength of 465 nm attached to a substrate 64a disposed on one side of the reaction unit 61, as shown in FIG. The light source part 62a and the photodiode light-receiving part 62b attached to the board | substrate 64b arrange | positioned at the other side surface side of the reaction part 61 are comprised. The LED light source unit 62a is an example of the “light source” in the present invention, and the photodiode light receiving unit 62b is an example of the “light detection unit” in the present invention. That is, in each reaction detection block 60a, two sets of one set of turbidity detection units 62 each including one LED light source unit 62a and one photodiode light receiving unit 62b are arranged. Accordingly, the five reaction detection blocks 60a are provided with a turbidity detection unit 62 including a total of ten sets of LED light source units 62a and photodiode light receiving units 62b. The LED light source unit 62a and the photodiode light receiving unit 62b corresponding to the LED light source unit 62a irradiate light having a diameter of about 1 mm from the LED light source unit 62a to the lower part of the detection cell 65 so that the light can be received by the photodiode light receiving unit 62b. Is arranged. The LED light source unit 62a and the photodiode light receiving unit 62b have functions of detecting the presence or absence of the detection cell 65 and detecting (monitoring) the turbidity of the liquid contained in the detection cell 65 in real time.

  Further, as shown in FIGS. 8 to 12, the detection cell 65 is a cell made of a heat-resistant transparent resin that can transmit light (for example, a crystalline olefin-based thermoplastic resin such as polymethylpentene (TPX)). It is configured by integrally combining two members, a member 66 and a lid member 67 made of heat-resistant resin (for example, high density polyethylene). The detection cell 65 is irradiated with an electron beam in a boxed state before shipment so that a degrading enzyme such as human saliva that may adhere in the manufacturing process of the detection cell 65 does not adversely affect gene amplification. It has been subjected. As shown in FIG. 10, the cell member 66 constituting the detection cell 65 is integrally formed with two cell portions 66a and two hook engaging holes 66b. Further, as shown in FIG. 11, the inner wall bottom 66c of the cell portion 66a of the cell member 66 can obtain a liquid level height that can be detected by the turbidity detection unit 62 even when the amount of liquid is small. It is formed in a round shape. Further, as shown in FIG. 12, the lid member 67 constituting the detection cell 65 has a cell having two lid parts 67a, two hook parts 67b, a knob part 67c, and two cell member mounting holes 67d. The member attaching portion 67e and the two connecting portions 67f are integrally formed. The lid portion 67a is an example of the “lid” in the present invention. The detection cell 65 is integrally assembled by inserting the two cell portions 66 a of the cell member 66 into the two cell member mounting holes 67 d of the lid member 67. When the lid portion 67a shown in FIG. 8 is opened and the lid portion 67a is closed as shown in FIG. 9, the two hook portions 67b of the lid member 67 are hook-engaged with the corresponding cell members 66, respectively. Engage with the hole 66b. Accordingly, the lid 67a is held in a closed state.

  As shown in FIGS. 7 and 13, the lid closing mechanism 63 is provided with a lid sealing arm 63 a for mounting the lid 67 a of the lid member 67 of the detection cell 65. The lid sealing arm 63a is integrally attached to the rotating member 63b. The rotating member 63b is attached to one end of the shaft 63c so as to be rotatable around the shaft 63c. As shown in FIGS. 13 and 14, a pulley 63d is attached to the other end of the shaft 63c. Further, a pulley 63e that is rotatably attached to the pulley 63d is provided at a predetermined interval. A belt 63f is attached between the pulley 63d and the pulley 63e. A vertical movement member 63g that moves up and down as the belt 63f moves up and down is attached to the belt 63f. As shown in FIG. 14, a tension spring 63h for urging the vertical movement member 63g downward is attached to the vertical movement member 63g. Further, as shown in FIG. 13, by pressing the vertical movement member 63g upward, the vertical movement member 63g is pressed upward to resist the urging force of the tension spring 63h (see FIG. 14). A member 63j is provided. The pressing member 63j is configured to move in the vertical direction using a stepping motor 63k and a slide screw 63i. In addition, a torque limiter 63l is provided between the stepping motor 63k and the slide screw 63i so as to idle when a torque more than necessary is applied. Further, as shown in FIG. 15, the pressing mechanism portion including the pressing member 63j, the stepping motor 63k, the torque limiter 63l, and the slide screw 63i is attached to the linear motion guide 63m so as to be movable in the X-axis direction. The pressing mechanism unit is moved in the X-axis direction between the five reaction detection blocks 60a by using the stepping motor 63n, the pulleys 63o and 63p, and the timing belt 63q.

  FIGS. 16 to 18 are schematic diagrams for explaining the lid closing operation of the measurement unit according to the first embodiment, and FIGS. 19 and 20 are graphs for explaining the gene amplification detection operation. Next, the operation of the nucleic acid detection device (gene amplification detection device) according to the first embodiment will be described with reference to FIGS. 1 to 4, 7 and 13 to 20. In the gene amplification detection apparatus according to the first embodiment, as described above, a cancer-derived gene (mRNA) present in a resected tissue in cancer surgery is amplified using the LAMP method, and the turbidity of the solution generated along with the amplification Detection is performed by measuring.

  First, as shown in FIG. 2 and FIG. 3, a sample container 22 containing a solubilized extract (sample) prepared by processing (homogenizing, filtering, diluting) the excised tissue in advance is used as a sample container setting base 21. To the sample container setting hole 21a. In addition, the primer reagent container 32a in which the primer reagent of CK19 (cytokeratin 19) is accommodated in the primer reagent container set hole 31a and the enzyme reagent container set hole 31b on the left side of the front and the enzyme reagent container 32b in which the enzyme reagent of CK19 is accommodated. Set. Also, a primer reagent container 32a in which a primer reagent of β-actin (β-actin) is accommodated in a primer reagent container set hole 31a and an enzyme reagent container set hole 31b on the right side of the front and an enzyme in which an enzyme reagent of β-actin is accommodated. The reagent container 32b is set. Further, two racks 42 each containing 36 disposable pipette tips 41 are fitted into the recesses (not shown) of the chip set section 40. In this case, as shown in FIGS. 2 and 3, the initial position of the arm portion 11 of the dispensing mechanism portion 10 is a position deviated from above the tip set portion 40. It is possible to fit two racks 42 (not shown). Further, the two cell portions 66a of the detection cell 65 are set in the two detection cell setting holes 61a of the reaction portion 61 of each reaction detection block 60a.

  Then, after registering measurement items, registering sample IDs, and the like using the keyboard 102a and mouse 102b of the data processing unit 102 shown in FIG. 1, the operation of the measurement unit 101 is started using the keyboard 102a or mouse 102b. .

  When the operation of the measurement unit 101 is started, first, after the arm unit 11 of the dispensing mechanism unit 10 is moved from the initial position to the chip set unit 40, the two syringe units of the dispensing mechanism unit 10 are moved in the chip set unit 40. 12 is moved downward. As a result, as shown in FIG. 4, the tips of the nozzle portions 12 a of the two syringe portions 12 are press-fitted into the upper openings of the two pipette tips 41, so that the pipettes are inserted into the tips of the nozzle portions 12 a of the two syringe portions 12. The chip 41 is automatically mounted. After the two syringe parts 12 are moved upward, the arm part 11 of the dispensing mechanism part 10 has two primers containing the CK19 and β-actin primer reagents set on the reagent container setting base 31. The reagent container 32a is moved in the X-axis direction upward. Then, by moving the two syringe parts 12 downward, the tips of the two pipette tips 41 attached to the nozzle parts 12a of the two syringe parts 12 are respectively CK19 in the two primer reagent containers 32a. And inserted into the surface of the β-actin primer reagent. Then, the CK19 and β-actin primer reagents in the two primer reagent containers 32a are aspirated by the pump part 12b of the syringe part 12. When the primer reagent is aspirated, the liquid level detection sensor 12d (see FIG. 4) detects that the tip of the pipette tip 41 made of conductive resin is in contact with the liquid level, and the pressure detection sensor 12e ( 4), the pressure at the time of suction by the pump unit 12b is detected. The liquid level detection sensor 12d and the pressure detection sensor 12e detect whether suction is being performed reliably.

  After the primer reagent is aspirated, after the two syringe parts 12 are moved upward, the arm part 11 of the dispensing mechanism part 10 is moved above the reaction detection block 60a located on the farthest side (the front side of the apparatus front side). Is done. In this case, the arm part 11 of the dispensing mechanism part 10 is moved so as not to pass above the second to fifth other reaction detection blocks 60a from the back. In the innermost reaction detection block 60a, the two syringe parts 12 are moved downward to detect the two pipette tips 41 attached to the nozzle parts 12a of the two syringe parts 12, respectively. The cell 65 is inserted into the two cell portions 66a. And using the pump part 12b of the syringe part 12, two primer reagents of CK19 and (beta) -actin are each discharged to the two cell parts 66a. At the time of this discharge, similarly to the above-described suction, the liquid level detection sensor 12d detects that the tip of the pipette tip 41 made of a conductive resin is in contact with the level of the discharged liquid and detects the pressure. The pressure at the time of discharge by the pump unit 12b is detected by the sensor 12e. These liquid level detection sensor 12d and pressure detection sensor 12e detect whether or not the ejection is reliably performed. It should be noted that the same detection by the liquid level detection sensor 12d and the pressure detection sensor 12e as described above is also performed when the following enzyme reagent and sample are aspirated and discharged.

  After the primer reagent is discharged, the two syringe parts 12 are moved upward, and then the arm part 11 of the dispensing mechanism part 10 is moved in the X-axis direction toward the upper part of the tip discarding part 50. In the tip discarding unit 50, the pipette tip 41 is discarded. Specifically, the pipette tip 41 is inserted into the two tip disposal holes 50a (see FIG. 3) of the tip disposal portion 50 by moving the two syringe portions 12 downward. In this state, when the arm portion 11 of the dispensing mechanism portion 10 is moved in the Y-axis direction, the pipette tip 41 is moved below the groove portion 50b. When the two syringe parts 12 are moved upward, the collar parts on the upper surface of the pipette tip 41 abut against the lower surfaces on both sides of the groove 50b and receive downward force from the lower surface. 41 is automatically detached from the nozzle part 12a of the two syringe parts 12. As a result, the pipette tip 41 is discarded in the tip discarding unit 50.

  Next, after the arm part 11 of the dispensing mechanism part 10 is moved again to the chip set part 40, the tip of the nozzle part 12a of the two syringe parts 12 is operated in the chip set part 40 by the same operation as described above. Two new pipette tips 41 are automatically mounted. The arm portion 11 of the dispensing mechanism portion 10 has an X-axis extending upward from the two enzyme reagent containers 32b in which the two enzyme reagents CK19 and β-actin set on the reagent container setting base 31 are accommodated. Moved in the direction. Thereafter, by moving the two syringe parts 12 downward, the two enzyme reagents CK19 and β-actin in the two enzyme reagent containers 32b are aspirated, and then the two syringe parts 12 are moved upward. Moved. Then, after the arm portion 11 of the dispensing mechanism portion 10 is moved above the innermost reaction detection block 60a, the two enzyme reagents CK19 and β-actin are respectively detected in the two cells of the detection cell 65. It is discharged to the part 66a. Also in this case, the arm part 11 of the dispensing mechanism part 10 is moved so as not to pass above the second to fifth other reaction detection blocks 60a from the back. After the enzyme reagent is discharged, the arm portion 11 of the dispensing mechanism portion 10 is moved above the tip discarding portion 50, and then the pipette tip 41 is discarded.

  Next, after the arm portion 11 of the dispensing mechanism portion 10 is moved again to the tip set portion 40, two new pipette tips 41 are automatically attached to the tips of the nozzle portions 12a of the two syringe portions 12. The The arm 11 of the dispensing mechanism 10 is moved in the X-axis direction toward the upper side of the sample container 22 in which the sample set on the sample container setting table 21 is accommodated, and then the sample in the sample container 22 Is sucked. Specifically, first, after one syringe part 12 located above one sample container 22 is moved downward and the sample is sucked, the one syringe part 12 is moved upward. Thereafter, the arm part 11 of the dispensing mechanism part 10 is moved in the Y-axis direction so that the other syringe part 12 is positioned above the same sample container 22. Then, after the other syringe part 12 is moved downward and the sample is sucked from the same sample container 22, the other syringe part 12 is moved upward. After that, after the arm part 11 of the dispensing mechanism part 10 is moved above the innermost reaction detection block 60a, the two syringe parts 12 are moved downward, and the two cell parts of the detection cell 65 are moved. In 66a, the same sample is discharged. Also in this case, the arm part 11 of the dispensing mechanism part 10 is moved so as not to pass above the second to fifth other reaction detection blocks 60a from the back.

  When the sample is discharged to the two cell portions 66a of the detection cell 65, the suction and discharge operations are repeated a plurality of times using the pumps 12b of the two syringe portions 12, so that the two cell portions 66a The contained CK19 and β-actin primer and enzyme reagents and the sample are agitated. At the time of dispensing the primer reagent, enzyme reagent, and sample, the liquid temperature in the detection cell 65 is maintained at about 20 ° C. using the Peltier module 61c shown in FIG. Thereafter, after the arm portion 11 of the dispensing mechanism portion 10 is moved above the tip discarding portion 50, the pipette tip 41 is discarded.

  Then, after the primer reagent, enzyme reagent, and sample are discharged into the cell portion 66a, the lid closing operation of the detection cell 65 is performed. Details of the lid closing operation will be described with reference to FIGS. 7 and 13 to 18. First, FIGS. 7 and 16 show a state in which the lid member 67 is opened immediately after the primer reagent, the enzyme reagent, and the sample are discharged into the cell portion 66a. From this state, when the stepping motor 63k shown in FIGS. 7 and 13 is rotationally driven in a predetermined direction, the slide screw 63i is rotated. Accordingly, the pressing member 63j is moved upward, so that the vertical movement member 63g (see FIG. 13) is moved upward against the urging force of the tension spring 63h (see FIG. 14). This upward movement of the vertically moving member 63g is converted into rotational motion of the shaft 63c via the belt 63f and the pulley 63d. As a result, the rotating member 63b attached to the shaft 63c is rotated in the direction of the arrow in FIGS. 7 and 16 with the shaft 63c as a fulcrum, so the lid sealing arm attached to the rotating member 63b. 63a is also rotated in the direction of the arrow with the shaft 63c as a fulcrum. Therefore, the lid portion 67a of the lid member 67 of the detection cell 65 placed on the lid sealing arm 63a is rotated toward the cell portion 66 side of the detection cell 65, and as shown in FIG. The lid portion 67a of the lid member 67 is closed with respect to 66a. Here, when a certain force is applied when closing the lid portion 67a with respect to the cell portion 66a, the torque limiter 63l shown in FIG. 13 rotates idly, so that an excessive force is applied to the lid portion 67a or the cell portion 66a. It can suppress adding. Thereby, it is possible to prevent the lid portion 67a or the cell portion 66a from being damaged or deformed during the lid closing operation. Note that once the lid portion 67a is closed, the hook portion 67b and the hook engagement hole 66b are engaged to maintain the closed state of the lid portion 67a, so that the lid portion 67a is opened again. Is prevented.

  Thereafter, when the stepping motor 63k shown in FIGS. 7 and 13 is rotationally driven in a direction opposite to the predetermined direction, the pressing member 63j is moved downward, so that the vertical movement member 63g (see FIG. 13). Is moved downward by the biasing force of the tension spring 63h (see FIG. 14). As a result, the shaft 63c is rotated in the opposite direction, so that the rotating member 63b attached to the shaft 63c is rotated in the direction of the arrow in FIG. 18 with the shaft 63c as a fulcrum. Thereby, the rotation member 63b and the lid sealing arm 63a are returned to the initial positions as shown in FIG. In this way, the lid closing operation is performed. The lid closing operation of the detection cell 65 is performed by the second reaction detection block 60a from the back after the primer reagent, enzyme reagent, and sample are discharged to the detection cell 65 of the reaction detection block 60a on the innermost side. This is performed before the primer reagent, enzyme reagent, and sample are discharged to the detection cell 65.

  After the above-described lid closing operation is completed, the liquid temperature in the detection cell 65 is heated from about 20 ° C. to about 65 ° C. by using the Peltier module 61c shown in FIG. To amplify the target gene (mRNA). And the cloudiness by magnesium pyrophosphate which is an amplification product produced | generated with amplification is detected by a turbidimetric method. Specifically, as shown in FIG. 18, light having a diameter of about 1 mm from the LED light source unit 62 a is passed through the light irradiation groove 61 b of the reaction unit 61 in the arrow B direction (see FIGS. 8 and 18). The cell portion 66a of the detection cell 65 during the amplification reaction is irradiated. The irradiated light is received by the photodiode light receiving unit 62b. Thereby, the liquid turbidity in the cell part 66a of the detection cell 65 during the amplification reaction is detected (monitored) in real time. As a result, in the data processing unit 102 (see FIG. 1), when the horizontal axis represents time and the vertical axis represents turbidity (OD: Optical Density), measurement data as shown in FIG. 19 is obtained. . Then, an amplification rise time which is a time until the copy number of the target gene (mRNA) in the sample increases rapidly from the measurement data is detected based on a change in turbidity. Then, the concentration of the target gene is calculated from the amplification rise time based on a calibration curve created in advance from the measurement result of the calibrator as shown in FIG. Here, the calibration curve shown in FIG. 20 is a curve with the amplification start-up time on the horizontal axis and the target gene concentration on the vertical axis. In general, the shorter the amplification start-up time, the higher the target gene concentration.

  In addition, a container containing a calibrator containing a target gene at a predetermined concentration that serves as a standard for creating a calibration curve, and a negative control for confirming that a gene that should not be amplified are not normally amplified The containers are set in the sample container setting hole 21a of the sample container setting table 21 at a predetermined frequency. For the calibrator and the negative control, operations similar to the above-described sample suction, discharge, and detection operations are performed. A calibration curve can be created by the detection operation of the calibrator, and it can be confirmed by the detection operation of the negative control that the gene that should not be amplified is not normally amplified.

  As described above, the target gene is detected in the reaction detection block 60a located on the innermost side. In parallel with the detection operation of the target gene after the lid closing operation in the reaction detection block 60a located on the innermost side, the primer reagent, enzyme reagent, and sample are dispensed in the reaction detection block 60a that is the second from the back. Operation, lid closing operation, and target gene detection operation are performed. Further, in parallel with the target gene detection operation after the lid closing operation in the second reaction detection block 60a from the back, the primer reagent, enzyme reagent, and sample dispensing operation are performed in the third reaction detection block 60a from the back. The lid closing operation and the target gene detection operation are performed. Thereafter, operations similar to the above are sequentially performed for the fourth and fifth reaction detection blocks 60a from the back. In the second to fifth reaction detection blocks 60a from the back, when the lid closing operation is performed, the stepping motor 63m shown in FIG. 15 is driven so that the pressing mechanism detects the innermost reaction. The lid 60 is sequentially moved from the block 60a to the second to fifth reaction detection blocks 60a from the back, and the lid closing operation is performed. The detection operation ends when the target gene detection operation of the fifth reaction detection block 60a ends. Thereafter, the grip portion 67c of the detection cell 65 is held and the five detection cells 65 are discarded.

  In the first embodiment, as described above, the lid closing mechanism 63 that closes the lid 67a of the detection cell 65 after the dispensing of the reagent and the sample to the detection cell 65 is provided, and the lid 67a is closed. In this state, by performing amplification of the target gene in the detection cell 65 and detection of the concentration of the target gene, it is possible to effectively prevent contamination of other samples and reagents by the amplified target gene.

  In the first embodiment, by using the detection cell 65 in which the lid portion 67a is integrally provided, the lid portion 67a does not fall on the other detection cells 65, so that the lid portion 67a is dropped. It is possible to prevent the samples and reagents of the other detection cells 65 from being contaminated due to the above.

  In the first embodiment, the lid closing operation by the lid closing mechanism 63 is performed after the primer reagent, the enzyme reagent, and the sample are discharged to the predetermined detection cell 65 and then to the next detection cell 65. By performing the discharge before the reagent and the sample are discharged, contamination (contamination) of the predetermined detection cell 65 can be surely prevented.

  In the first embodiment, as described above, the nozzle portion 12a of the dispensing mechanism portion 10 is provided with the nozzle portion 12a to which the pipette tip 41 is detachably attached to the tip thereof, thereby the nozzle of the syringe portion 12 Contamination can be prevented by exchanging the pipette tip 41 detachably attached to the part 12a for each sample or reagent.

  Further, in the first embodiment, as described above, the turbidity detection unit 62 that detects the liquid turbidity in the detection cell 65 is easily configured by the LED light source unit 62a and the photodiode light receiving unit 62b. The presence of the amplified target gene in the detection cell 65 can be detected. In this case, more accurate detection of liquid turbidity can be performed by detecting (monitoring) the liquid turbidity in the detection cell 65 during amplification reaction in real time using the LED light source unit 62a and the photodiode light receiving unit 62b. It can be carried out. Thereby, since the detection accuracy of the amplification rise time can be improved, the detection accuracy of the target gene concentration can be improved.

  In the first embodiment, as described above, the target gene can be efficiently amplified by performing the amplification of the target gene by the reaction unit 61 using the LAMP method, which is a direct gene amplification method for a short time. Therefore, the time required for detecting the target gene can be shortened. That is, in the first embodiment, by using the LAMP method, it is possible to perform from sample setting to reaction detection in a short time of about 30 minutes.

  In the first embodiment, as described above, the amplification of the target gene by the reaction unit 61 and the detection of the target gene by the turbidity detection unit 62 are performed at the same position, as described above. And the detection of the target gene by the turbidity detection unit 62 can be simplified, and the configuration of the apparatus can be simplified and the detection container does not move from the amplification position to the detection position. , Processing time can be shortened.

  In the first embodiment, the target gene amplification by the reaction unit 61 and the target gene detection by the turbidity detection unit 62 are performed in parallel, thereby reducing the time for target gene amplification and detection processing. be able to.

  Further, in the first embodiment, as described above, the lid closing operation by the lid closing mechanism 63 is performed only once for one detection cell 65, and the lid 67a is not subsequently opened. Since there is no step of opening the lid 67a, the processing can be speeded up.

(Second Embodiment)
FIG. 21 is a perspective view showing the overall configuration of the measurement unit of the nucleic acid detection device according to the second embodiment of the present invention. FIG. 22 is a schematic plan view of FIG. 23 to 46 show details of the components of the measurement unit of the nucleic acid detection device according to the second embodiment shown in FIG. 21, and FIGS. 47 and 48 show the second embodiment shown in FIG. It is a top view for demonstrating operation | movement of the measurement part of the nucleic acid detection apparatus by form. Referring to FIGS. 21, 22, 35, and 41, in the second embodiment, unlike the first embodiment, the condensed water accumulated in the sample container setting unit 420 and the sample container setting unit 430 is discharged. Condensed water discharge mechanism, tip discarding section 450 including tip disposal bag 452, and dripping removal for removing dripping generated at the tip of pipette tip 41 when a sample is discharged to cell portion 66a of detection cell 65 The member 410 is provided in the measurement unit 401, and the structure of the lid closing mechanism unit 461 of the reaction detection unit 460 is different from that of the first embodiment. Moreover, in 2nd Embodiment, arrangement | positioning of the mechanism which moves the dispensing mechanism part 10 within XY plane is different from 1st Embodiment. The remaining structure of the second embodiment is basically the same as that of the first embodiment. The operations other than the chip waste bag setting operation, the dripping removal operation, and the lid closing operation of the second embodiment are basically the same as those of the first embodiment. Hereinafter, the second embodiment will be described in detail.

  First, with reference to FIGS. 21 to 33, a sample container setting unit 420, a sample container setting unit 430, and their condensed water discharge mechanisms according to the second embodiment will be described. In the second embodiment, as shown in FIGS. 21 to 25, the sample container setting base 422 is detachably fitted in the recess 421 of the sample container setting section 420. As shown in FIGS. 26 to 29, the sample container set base 422 includes an aluminum base 423, a transparent resin plate 424, and a heat insulating material 425. As shown in FIGS. 26 and 29, the aluminum base 423 has five sample container setting holes 423a. Sample containers 22 (see FIG. 21) similar to those in the first embodiment are set in the five sample container setting holes 423a. The resin plate 424 constituting the sample container setting table 422 has two gripping portions 424a and five through holes 424b formed at positions corresponding to the five sample container setting holes 423a of the base 423. Yes. As shown in FIGS. 28 and 29, the heat insulating material 425 is disposed between the plate 424 and the base 423 so as to surround the five sample container setting holes 423a.

  As shown in FIGS. 23 and 25, a reagent container setting base 432 is detachably fitted in the recess 431 of the reagent container setting section 430. As shown in FIGS. 30 to 33, the reagent container set base 432 includes an aluminum base 433, a transparent resin plate 434, and a heat insulating material 435. As shown in FIGS. 30 and 33, the aluminum base 433 has two primer reagent container setting holes 433a and one enzyme reagent container setting hole 433b. As shown in FIGS. 21 and 22, the two primer reagent container setting holes 433a are provided at predetermined intervals along the Y-axis direction, and the enzyme reagent container setting hole 433b is provided only on the left side of the front surface. Is provided. An enzyme reagent container 436 containing an enzyme reagent common to cytokeratin 19 (CK19) and β-actin is disposed in the enzyme reagent container set hole 433b. The primer reagent container 32a disposed in the two primer reagent container setting holes 433a is the same as that in the first embodiment. As shown in FIGS. 30 and 33, the resin plate 434 constituting the reagent container set base 432 includes two gripping portions 434a, two primer reagent container set holes 433a of the base 433, and one enzyme reagent container set. And three through-holes 434b formed at positions corresponding to the holes 433b. As shown in FIGS. 31 to 33, the heat insulating material 435 is disposed between the plate 434 and the base 433 so as to surround the two primer reagent container setting holes 433a and the one enzyme reagent container setting hole 433b.

  As shown in FIG. 25, a plurality of water collecting grooves 440 are formed on the bottom surface of the concave portion 421 of the sample container setting portion 420 and the bottom surface of the concave portion 431 of the reagent container setting portion 430. The plurality of water collecting grooves 440 are provided so as to penetrate from the inner side surfaces of the recesses 421 and 431 to the outer side surfaces of the sample container setting unit 420 and the reagent container setting unit 430. In addition, drain pipes 442 that are inclined downward toward the tip are connected to the ends of the water collecting grooves 440 located on the outer surface portions of the sample container setting section 420 and the reagent container setting section 430, respectively. As shown in FIGS. 23 and 25, a bowl-shaped drainage groove 441 extending along the X-axis direction is formed below the distal end portion of the drainage pipe 442. As shown in FIGS. 21 and 24, this bowl-shaped drainage groove 441 has a shape that inclines downward as it approaches the front side of the apparatus. The water collecting groove 440, the drain groove 441, and the drain pipe 442 constitute a dew condensation water discharge mechanism. This dew condensation water discharging mechanism collects the dew condensation water generated on the surfaces of the aluminum base 423 of the sample container set base 422 and the aluminum base 433 of the reagent container set base 432, and the water collecting groove 440, the drain pipe 442 and the drain groove 441. Is provided to discharge the sample container set unit 420 and the reagent container set unit 430 to the outside. In addition, as shown in FIG. 21, the dew condensation water storage container 500 is installed under the front-end | tip part of the drain groove 441. As shown in FIG.

  Next, the chip disposal unit 450 according to the second embodiment will be described with reference to FIGS. 21, 22, and 34 to 40. The chip disposal unit 450 shown in FIGS. 21 and 22 includes a box-shaped storage unit 451, a chip disposal bag 452 disposed in the storage unit 451, and a chip disposal bag, as shown in FIGS. A bag set base 453 for setting 452, a bag detection sensor 454, and a discard hole forming portion 455 are provided. As shown in FIGS. 35 and 40, the chip waste bag 452 is formed with a chuck 452a for enabling the chip waste bag 452 to be opened and closed.

  Further, as shown in FIGS. 38 and 39, the bag setting table 453 includes an L-shaped main body 453a, a bag holding member 453b rotatably attached to the upper portion of the main body 453a, and a main body 453a. It is comprised from the handle 453c attached to the outer surface. A resin-made support base 453e is attached to the bottom surface portion 453d of the L-shaped main body portion 453a. As shown in FIG. 35, the resin support base 453e is formed in a shape along the bottom of the concave outer surface of the chip disposal bag 452, and has two chamfers for making it difficult to damage the chip disposal bag 452. Part 453f. The bag holding member 453b has a through hole 453g and a pair of overhang portions 453h extending downward from a predetermined position facing each other across the through hole 453g. As shown in FIG. 35, the pair of overhang portions 453h is configured so that when the chip disposal bag 452 is set on the bag setting table 453, the upper portion of the chip disposal bag 452 can be maintained open. It is comprised so that it may contact | abut to the inner surface side. In addition, a U-shaped notch 453i is formed in the pair of overhang portions 453h. As shown in FIG. 35, the chip disposal bag 452 is sandwiched from above and below by a bottom surface portion 453d of a main body portion 453a to which a support base 453e is attached and a bag holding member 453b having an overhang portion 453h. It has been fixed.

  Further, as shown in FIGS. 36 and 37, the bag detection sensor 454 is attached to the outer surface of the box-shaped storage unit 451 so as to face each other with the storage unit 451 interposed therebetween. The bag detection sensor 454 is provided to detect whether or not the chip disposal bag 452 is normally set on the bag setting table 453 when the bag setting table 453 is fitted in the storage unit 451. Moreover, the bag detection sensor 454 has a rotating member 454b to which a pulley 454a is attached at a predetermined position. The rotating member 454b and the pulley 454a are provided so as to protrude into the storage portion 451 through a through hole (not shown) provided in the side surface of the storage portion 451. Further, the pulley 454a of the rotating member 454b is disposed at a position corresponding to the position of the notch 453i of the bag set base 453 in a state where the bag set base 453 is fitted in the storage portion 451.

  Next, with reference to FIG. 21 and FIG. 41, the dripping removal member 410 by 2nd Embodiment is demonstrated. In the second embodiment, as shown in FIGS. 21 and 41, the dispensing mechanism unit 10 can protrude into the X-axis direction (the direction of arrow M in FIG. 41) below the dispensing mechanism unit 10. A liquid dripping removal member 410 is provided. The dripping removal member 410 is provided to receive dripping dropped from the tip of the pipette tip 41 when a sample is discharged to the cell portion 66a of the detection cell 65. As shown in FIG. 41, the dripping removal member 410 is provided with a concave mounting portion 411, and a resin tray member 412 is detachably fitted into the mounting portion 411. The tray member 412 has two concave portions 412a and a grip portion 412b formed between the two concave portions 412a. The two concave portions 412a of the tray member 412 are respectively recessed below the two pipette tips 41 attached to the two syringe portions 12 of the dispensing mechanism portion 10 when the dripping removal member 410 protrudes. Are provided at a predetermined interval so as to be positioned.

Next, the detailed structure of the lid closing mechanism 461 of the reaction detection unit 460 according to the second embodiment will be described with reference to FIGS. 21, 22, and 42 to 44. The reaction detection unit 460 is an example of the “amplification unit” and the “detection unit” in the present invention . The cover closed mechanism 461, FIG. 21, as shown in FIGS. 22 and 42, the lid support member 461a for mounting the cover portion 67a of the lid member 67 of the detection cell 65 is provided. The one side end portion of the lid support members 461a of this, as shown in FIGS. 22 and 42, the rotating member 461c is attached via a support shaft 461b. Further, a detection piece 461e is attached to the other end portion of the lid side support member 461a via a support shaft 461d. Further, a light transmission type sensor 462 is provided in the vicinity of the detection piece 461e. The sensor 462 is provided to detect whether or not the lid member 67 of the detection cell 65 is closed by detecting that the detection piece 461e has reached a predetermined rotation position. Further, as shown in FIG. 43, the rotating member 461c attached to one end of the lid-side support member 461a is urged downward by a tension spring 461f. This tension spring 461f is in the state of I in FIG. 43 (the state where the lid member 67 of the detection cell 65 is closed) or the state of J (the lid member 67 of the detection cell 65 is not closed) with the support shaft 461b as a fulcrum. The rotating member 461c is urged downward in either state.

  Further, as shown in FIGS. 42 and 43, two protrusions 461g and 461h are formed at predetermined positions on the outer surface of the rotating member 461c. In the state of J in FIG. 43 (a state where the lid member 67 of the detection cell 65 is not closed), the protruding portion 461g is positioned at a predetermined height on the upper side, and the protruding portion 461h is on the lower side. Located at a predetermined height. Further, when the rotating member 461c is in the state I of FIG. 42 (a state where the lid member 67 of the detection cell 65 is closed), the protruding portion 461h is positioned at a predetermined height on the upper side, and the protruding portion 461g is on the lower side. Located at a predetermined height. In each reaction detection block 460a, as shown in FIGS. 42 and 43, a sensor (micro switch) for detecting whether or not the lid member 67 of the detection cell 65 is set on the lid-side support member 461a. 463 is provided. The sensor 463 is provided at a position where the knob portion 67c of the lid member 67 contacts when the lid member 67 of the detection cell 65 is set on the lid side support member 461a.

  Further, as shown in FIGS. 22, 42 and 44, pulleys 461j and 461k that rotate with a predetermined interval in the X-axis direction are arranged on the side where the rotating member 461c is installed. A stepping motor 461i for rotationally driving the pulley 461j is provided below the pulley 461j. A belt 461l is attached between the pulley 461j and the pulley 461k. An electromagnetic valve attachment member 461m is attached to the belt 461l by a belt attachment portion 461n. The electromagnetic valve attachment member 461m is attached to a slider 461t (see FIG. 44) attached to the rail portion 461s of the linear guide so as to be slidable in the X-axis direction. An electromagnetic valve 461o is attached to the electromagnetic valve attachment member 461m. A pressing member 461r having a flat pressing portion 461q projecting toward the rotating member 461c is attached to the end of the movable shaft 461p of the electromagnetic valve 461o on the rotating member 461c side. The pressing portion 461q of the pressing member 461r is moved in the direction of arrow G in FIG. 44 by the electromagnetic valve 461o, and is moved in the direction of arrow F in FIG. 42 by the belt 461l. The protrusion 461g or 461h can be pressed. In this case, the pressing member 461q is configured to contact only the upper protruding portion 461g (461h) of the two protruding portions 461g and 461h of the rotating member 461c. As shown in FIG. 21, the lid-side support member 461a is arranged so that the lid portion 67a is inclined 45 degrees from the horizontal direction when the lid portion 67a is opened and the detection cell 65 is set in the reaction portion 61. . Accordingly, the user can easily set and remove the detection cell 65 from the reaction unit 61. The inclination of the lid portion 67a is most preferably about 45 degrees, but if it is inclined within a range of 30 degrees to 60 degrees, it is easy to set and remove.

  Further, as shown in FIGS. 21, 22, 45 and 46, the lid pressing member 464a is provided in the Z-axis direction (vertical direction) via the linear motion guide 464b on the arm portion 11 of the dispensing mechanism portion 10. ) Is movably attached. The lid pressing member 464a is an example of the “pressing member” in the present invention. As shown in FIG. 46, the lid pressing member 464a is urged upward by a tension spring 464c. Further, as shown in FIGS. 22, 45 and 46, the lid pressing member 464 a is pressed downward on the back surface of the frame 470 that supports the dispensing mechanism unit 10, so that the lid pressing member 464 a is pressed. A pressing member driving unit 465 is provided for moving downward against the urging force of the tension spring 464c (see FIG. 46). The pressing member drive unit 465 includes a stepping motor 465a, pulleys 465b and 465c, a belt 465d, a torque limiter 465e, a slide screw 465f, a linear motion guide 465g, and a vertical motion member 465h. Specifically, a stepping motor 465a and a pulley 465b that rotates in synchronization with the rotation of the stepping motor 465a are provided on the back surface of the frame 470 that supports the dispensing mechanism unit 10. A pulley 465c is provided at a predetermined interval from the pulley 465b. A belt 465d is attached between the pulley 465b and the pulley 465c. Further, a sliding screw 465f is connected to the shaft portion of the pulley 465c via a torque limiter 465e that idles when an excessive torque is applied. A vertical movement member 465h is attached to the slide screw 465f so as to be movable in the vertical direction. Further, the vertical movement member 465h is attached to the frame 470 so as to be movable in the vertical direction (Z-axis direction) via a linear motion guide 465g. The vertical movement member 465h is configured to move in the vertical direction as the slide screw 465f rotates.

  Next, the operation of the nucleic acid detection device (gene amplification detection device) according to the second embodiment will be described with reference to FIGS. Here, operations other than the chip waste bag setting operation, the dripping removal operation, and the lid closing operation of the second embodiment are basically the same as those of the first embodiment described above. However, in the second embodiment, unlike the first embodiment, only one enzyme reagent container 436 can be set. Therefore, the two syringe parts 12 of the dispensing mechanism part 10 are provided in the enzyme reagent container 436. It is necessary to change the operation of sucking the sample to the same operation as the operation of sucking the sample in the sample container 22 in the first embodiment.

  First, with reference to FIGS. 34 to 40, the operation of setting the chip disposal bag 452 on the bag setting table 453 will be described. This operation is performed by the user before the measurement operation by the measurement unit 401 is started. First, the handle 453c is grasped and the bag set base 453 is pulled out from the storage portion 451, and the bag holding member 453b of the bag set base 453 is rotated about 90 degrees in the direction of arrow B in FIG. 38 from the state shown in FIG. As a result, the state shown in FIG. 39 is obtained. Then, with the chip waste bag 452 (see FIG. 40) opened, the bottom of the chip waste bag 452 is fitted into the support base 453e of the bag setting base 453. Then, by rotating the bag holding member 453b of the bag setting table 453 by about 90 degrees in the direction of arrow C in FIG. 39, the chip disposal bag 452 is set on the bag setting table 453 as shown in FIG. become. After the chip waste bag 452 is set on the bag setting table 453 as described above, the handle 453c shown in FIG. 35 is held and the bag setting table 453 is fitted into the storage portion 451 from the direction of arrow D shown in FIG. In this case, since the overhanging portion 453h (see FIG. 35) of the bag setting table 453 is in contact with the inner surface side of the chip disposal bag 452, the outer surface of the chip disposal bag 452 is the bag detection sensor 454 (FIGS. 36 and 37). (See) pulley 454a. As a result, the pulley 454a receives force from the outer surface of the chip disposal bag 452 in the direction of the bag detection sensor 454 (the direction of arrow E in FIGS. 36 and 37), so that the rotating member 454b of the bag detection sensor 454 It turns to the direction of the bag detection sensor 454, and will be in the state shown in FIG. Accordingly, since the bag detection sensor 454 is turned on, it is determined that the chip disposal bag 452 is normally set.

  In a state where the chip disposal bag 452 is not set normally, or in a state where the chip disposal bag 452 is not set normally, such as the overhanging portion 453h of the bag setting table 453 is located outside the chip disposal bag 452, When the bag set base 453 is fitted into the storage portion 451, the pulley 454 a of the rotating member 454 b enters the notch 453 i of the bag set base 453. In this case, the rotation member 454b does not rotate in the direction of the bag detection sensor 454 (the direction of arrow E in FIGS. 36 and 37). In this case, since the bag detection sensor 454 is not turned on, it is determined that the chip disposal bag 452 is not set on the bag setting table 453.

  After the chip waste bag 452 is normally set as described above, the sample container 22, the primer reagent container 32a, and the enzyme reagent container 436 are set as in the first embodiment. Then, the operation of the measurement unit 401 is started. Then, the mounting operation of the pipette tip 41, the suction and discharge operation of the sample, the primer reagent and the enzyme reagent, the chip discarding operation, the lid closing operation after the sample discharge and the amplification detection operation are performed. In the chip discarding operation according to the second embodiment, the pipette tip 41 discarded in the chip discarding hole 455a of the discarding hole forming unit 455 is discarded by the chip setting table 453 positioned below the discarding hole forming unit 455. The bag 452 is held inside. Then, after the target gene detection operation of the five reaction detection blocks 460a is completed, the user takes out the bag setting table 453 on which the chip disposal bag 452 is set from the storage unit 451. Then, after the bag holding member 453b is rotated upward from the state shown in FIG. 35, the chuck 452a of the chip disposal bag 452 is closed. Then, the chip disposal bag 452 is discarded in a predetermined disposal facility.

  In the second embodiment, unlike the first embodiment, the dripping removal operation is performed during the sample discharging operation. Specifically, as shown in FIG. 41 and FIG. 47, immediately after the sample discharging operation, an electromagnetic valve (not shown) provided in the dispensing mechanism unit 10 is driven, whereby the dripping removal member 410 is driven. Protrudes in the X-axis direction (the direction of arrow M in FIG. 41) with respect to the two pipette tips 41. And when the residual liquid of a sample falls from the front-end | tip of two pipette tips 41, before the residual liquid falls in the measurement part 401, two recessed parts of the saucer member 412 fitted by the dripping removal member 410 were carried out. Received in 412a. Thereby, the residual liquid of the sample generated at the tips of the two pipette tips 41 is removed.

  Further, in the lid closing operation after discharging the sample according to the second embodiment, unlike the first embodiment, the lid side support member 461a is rotated by the electromagnetic valve mounting member 461m (see FIGS. 42 to 44). Thereafter, a lid pressing operation is performed on the lid-side support member 461a of the lid pressing member 464a. The details of the lid closing operation according to the second embodiment will be described below with reference to FIGS. 22, 42 to 46 and 48.

  As the rotation operation of the lid-side support member 461a by the electromagnetic valve mounting member 461m, first, from the state in which the lid portion 67a immediately after the primer reagent, enzyme reagent and sample are discharged into the cell portion 66a is opened, The stepping motor 461i shown in FIGS. 22, 42 and 44 is rotationally driven in a predetermined direction. As a result, the electromagnetic valve mounting member 461m is moved toward the front of the apparatus (in the direction of arrow F in FIG. 42) via the pulley 461j and the belt 461l. At this time, before the electromagnetic valve mounting member 461m moves to the position of the reaction detection block 460a where the lid is closed, the electromagnetic valve 461o is energized and has a pressing portion 461q attached to the movable shaft 461p of the electromagnetic valve 461o. The member 461r is moved in the direction of the arrow G in FIGS. As a result, the pressing portion 461q of the pressing member 461r abuts on the protruding portion 461g located on the upper side of the rotating member 461c, and presses the protruding portion 461g in the direction of arrow H in FIG. Therefore, the rotation member 461c is rotated in the direction of the arrow H in FIG. 43 against the urging force of the tension spring 461f with the support shaft 461b as a fulcrum. At this time, the lid-side support member 461a attached to the rotation member 461c via the support shaft 461b is also rotated in the direction of arrow H in FIG. 43, so that the detection cell placed on the lid-side support member 461a. The lid portion 67 a of the 65 lid member 67 is rotated to the cell member 66 side of the detection cell 65. In this state, a lid pressing operation is performed on the lid-side support member 461a by the lid pressing member 464a.

  As the lid pressing operation for the lid-side support member 461a by the lid pressing member 464a, first, the frame 470 supporting the dispensing mechanism unit 10 is moved in the X-axis direction from the origin position shown in FIG. The mechanism unit 10 is moved in the Y-axis direction with respect to the frame 470. As a result, as shown in FIGS. 44 to 46 and 48, the lid pressing member 464a provided in the dispensing mechanism unit 10 is located above the lid-side support member 461a rotated by the rotation operation, and , It is moved to a position directly below the vertical movement member 465h provided on the frame 470. In this state, when the stepping motor 465a shown in FIG. 45 is rotationally driven in a predetermined direction, the pulley 465c rotates via the pulley 465b and the belt 465d, so that the slide screw 465f connected to the pulley 465c is Rotate. Accordingly, the vertical movement member 465h is moved downward (in the direction of the arrow L in FIGS. 45 and 46), so that the lid pressing member 464a is moved downward (FIG. 44 to FIG. 44) against the urging force of the tension spring 464c. It is moved in the direction of arrow K in FIG. Accordingly, the lid-side support member 461a is pressed from above by the lid pressing member 464a, so that the cell portion 66a of the detection cell 65 is reliably closed by the lid portion 67a. In this state, the detection piece 461e attached to the lid-side support member 461a via the support shaft 461d reaches the position detected by the sensor 462, so that the sensor 462 is turned on. As a result, it is determined that the lid closing operation has been normally performed. Thereby, the lid pressing operation for the lid-side support member 461a of the lid pressing member 464a is completed. When the vertical movement member 465h and the lid pressing member 464a press the lid side support member 461a from above, if a force exceeding a certain level is applied, the torque limiter 465e shown in FIG. It is suppressed that excessive force is added to 67a and the cell part 66a.

  Thereafter, the stepping motor 465a is rotationally driven in a direction opposite to the predetermined direction described above, whereby the vertical movement member 465h is moved upward (the direction opposite to the direction of arrow L in FIGS. 45 and 46). Therefore, the lid pressing member 464a is moved upward (the direction opposite to the direction of the arrow K in FIGS. 44 to 46) by the urging force of the tension spring 464c. Further, when the stepping motor 461i shown in FIGS. 22, 42 and 44 is driven to rotate in a direction opposite to the predetermined direction, the electromagnetic valve mounting member 461m is moved in the rear direction of the device (in the direction of the arrow F in FIG. 42). Is moved in the opposite direction). At this time, if the electromagnetic valve 461o is in an energized state, the pressing portion 461q of the pressing member 461r moves the protruding portion 461h located above the two protruding portions 461g and 461h of the rotating member 461c in the direction of arrow H in FIG. Press in the opposite direction. Thereby, the rotation member 461c is rotated in the direction opposite to the direction of the arrow H in FIG. 43 against the urging force of the tension spring 461f with the support shaft 461b as a fulcrum. At this time, the lid side support member 461a is also rotated in the direction opposite to the direction of the arrow G in FIG. 42 via the support shaft 461b, and returns to the initial position (state J in FIG. 43). In this way, a series of lid closing operations are performed. The operation of returning the lid-side support member 461a to the initial position (state J in FIG. 43) is performed after the sample amplification and detection operations similar to those in the first embodiment are completed.

  In the second embodiment, as described above, the lid closing mechanism 461 is configured such that the lid side support member 461a that rotates the lid 67a of the detection cell 65 to the lid closed position, and the lid side support member 461a from above. An upper direction by the lid pressing member 464a includes a lid pressing member 464a that applies a pressing force from above to the lid portion 67a by pressing downward to complete the lid closing operation of the lid portion 67a. By the lid closing operation by the pressing force from the first embodiment, the lid closing operation can be performed more reliably as compared with the first embodiment in which the lid closing operation is performed only by the rotation operation.

  Further, in the second embodiment, as described above, the condensation generated in the sample container setting unit 420 and the reagent container setting unit 430 is provided by providing the condensed water discharge mechanism in the sample container setting unit 420 and the reagent container setting unit 430. It is possible to suppress water from accumulating in the sample container setting unit 420 and the reagent container setting unit 430.

  Moreover, in 2nd Embodiment, drainage of dew condensation water is efficiently performed by forming the drainage groove | channel 441 of a dew condensation water discharge mechanism in the shape which inclines below as it goes to an apparatus front near side as mentioned above. Can do.

  Further, in the second embodiment, as described above, by disposing the tip disposal bag 452 in the tip disposal section 450 so that the discarded pipette tip 41 is stored in the tip disposal bag 452, The user can carry the pipette tip 41 out of the analyzer without touching the discarded pipette tip 41 using the tip waste bag 452.

  In the second embodiment, as described above, by providing the two chamfered portions 453f on the support base 453e of the bag setting table 453, when the chip disposal bag 452 is set on the bag setting table 453, the chip disposal bag 452 can be prevented from being damaged.

  Further, in the second embodiment, as described above, by providing the liquid dripping removal member 410 in the dispensing mechanism unit 10, the sample drops from the tip of the pipette tip 41 when the sample is discharged to the cell part 66a of the detection cell 65. Therefore, it is possible to suppress the residual liquid of the sample from adhering to the measurement unit 401.

  The remaining effects of the second embodiment are similar to those of the aforementioned first embodiment.

  The embodiment disclosed this time should be considered as illustrative in all points and not restrictive. The scope of the present invention is shown not by the above description of the embodiments but by the scope of claims for patent, and further includes all modifications within the meaning and scope equivalent to the scope of claims for patent.

  For example, in the above embodiment, the nucleic acid detection device of the present invention is applied to a gene amplification detection device that amplifies a target gene by the LAMP method. However, the present invention is not limited to this, and the target gene is converted to a polymerase chain reaction. You may apply to the gene amplification detection apparatus amplified by the method (PCR method) or the ligase chain reaction method (LCR method). Further, the nucleic acid detection device of the present invention may be applied to a nucleic acid detection device other than the gene amplification detection device.

  In the first and second embodiments, the example in which the sample containers 22 are arranged in one row in the X-axis direction on the sample container setting bases 21 and 422 has been shown. The sample containers 22 may be arranged on the bases 21 and 422 in two rows in the X-axis direction. In this case, if the same sample is accommodated in the sample container 22 adjacent in the Y-axis direction, the same sample can be sucked at the same time by the two syringe parts 12, and therefore the processing can be further speeded up. .

  In the above embodiment, an example is shown in which the detection cell 65 is integrally provided with the lid by integrally combining the two members of the cell member 66 and the lid member 67. However, the detection cell provided with the cover integrally may be formed by one member.

  Further, in the above embodiment, both the dispensing of the reagent and the dispensing of the sample are performed by the single dispensing mechanism unit 10, but the present invention is not limited to this, and the dispensing mechanism that dispenses the reagent. And a dispensing mechanism for dispensing the sample may be provided separately.

  Moreover, in the said embodiment, although the turbidity detection part 62 which detects the liquid turbidity in the detection cell 65 was comprised from the LED light source part 62a and the photodiode light-receiving part 62b, this invention showed to this. Not limited to this, a turbidity detection unit composed of detection means other than the LED light source unit and the photodiode light receiving unit may be used. For example, the turbidity detection unit may be configured by a light source unit in which an optical fiber is connected to a lamp light source and a light receiving unit (light detection unit) that can receive light from the optical fiber of the light source unit.

  Moreover, in the said embodiment, although the example which detects a target gene by detecting the cloudiness by the amplification product (magnesium pyrophosphate) in the detection cell 65 by the turbidity detection part 62 was shown, this invention is shown to this. The target gene may be detected by detecting a reagent that binds to the amplification product of the target gene by a predetermined detection means. As a reagent in this case, for example, ethidium bromide or TaqMan probe can be used.

  Further, in the above embodiment, the detection cell 65 and the dispensing mechanism unit 10 are used in duplicate, but a detection cell or a dispensing mechanism unit including a single cell unit or a single syringe unit is used. May be.

In the second embodiment, the condensed water discharge mechanism is provided under both the sample container set base 422 and the reagent container set base 432. However, the present invention is not limited to this, and only under the sample container set base 422, Alternatively, a dew condensation water discharge mechanism may be provided only under the reagent container set base 432.
In the first and second embodiments, the pipette tips 41 discarded in the tip discarding units 50 and 450 are discarded as they are, but they may be washed and reused.

It is the perspective view which showed the whole structure of the nucleic acid detection apparatus (gene amplification detection apparatus) by 1st Embodiment of this invention, and its peripheral device. It is the perspective view which showed the whole structure of the measurement part of the nucleic acid detection apparatus shown in FIG. FIG. 3 is a schematic plan view of a measurement unit of the nucleic acid detection device shown in FIG. 2. It is the schematic which showed the structure of the syringe part in the measurement part of the nucleic acid detection apparatus shown in FIG. It is sectional drawing which showed the structure of the pipette chip | tip used for the nucleic acid detection apparatus shown in FIG. It is the perspective view which showed the storage state of the rack which accommodates the pipette chip | tip used for the nucleic acid detection apparatus shown in FIG. It is an expansion perspective view of the reaction detection part in the measurement part of the nucleic acid detection apparatus shown in FIG. FIG. 3 is a perspective view showing a state in which a lid of a detection cell used in the measurement unit of the nucleic acid detection device shown in FIG. 2 is opened. It is the perspective view which looked at the state which the lid | cover of the detection cell used for the measurement part of the nucleic acid detection apparatus shown in FIG. 2 closed from the arrow A direction of FIG. It is the perspective view which showed the cell member which comprises the detection cell shown in FIG. It is sectional drawing of the cell member which comprises the detection cell shown in FIG. It is the perspective view which showed the cover member which comprises the detection cell shown in FIG. It is the front view which showed the lid-closing mechanism part of the reaction detection part in the measurement part of the nucleic acid detection apparatus shown in FIG. It is a partial side view of the lid closing mechanism part shown in FIG. It is a top view of the lid closing mechanism part shown in FIG. It is the schematic for demonstrating operation | movement of the lid closing mechanism part shown in FIG. It is the schematic for demonstrating operation | movement of the lid closing mechanism part shown in FIG. It is the schematic for demonstrating operation | movement of the lid closing mechanism part shown in FIG. It is the graph which showed the relationship between the time measured by the nucleic acid detection apparatus shown in FIG. 2, and turbidity. 3 is a graph on which a calibration curve showing the relationship between the amplification rise time used in the nucleic acid detection apparatus shown in FIG. 2 and the target gene concentration is drawn. It is the perspective view which showed the whole structure of the measurement part of the nucleic acid detection apparatus by 2nd Embodiment of this invention. It is the plane schematic diagram of the measurement part of the nucleic acid detection apparatus shown in FIG. It is the top view which showed the sample container setting part and the reagent container setting part in the measurement part of the nucleic acid detection apparatus shown in FIG. It is a right view of the sample container setting part and reagent container setting part which were shown in FIG. It is the top view which showed the state which removed the sample container set stand and the reagent container set stand from the state shown in FIG. It is a top view of the sample container setting stand which comprises the sample container setting part shown in FIG. It is a front view of the sample container setting stand shown in FIG. It is a right view of the sample container setting stand shown in FIG. It is sectional drawing along the 500-500 line | wire in FIG. FIG. 24 is a plan view of a reagent container setting table constituting the reagent container setting unit shown in FIG. 23. It is a front view of the reagent container setting stand shown in FIG. FIG. 31 is a right side view of the reagent container setting table shown in FIG. 30. It is sectional drawing along the 600-600 line in FIG. It is the schematic which showed the state which inserted the bag set stand in the accommodating part in the measurement part of the nucleic acid detection apparatus shown in FIG. It is the perspective view which showed the state which set the chip disposal bag to the bag set stand in the measurement part of the nucleic acid detection apparatus shown in FIG. It is the top view which showed the accommodating part in the measurement part of the nucleic acid detection apparatus shown in FIG. It is a left view of the accommodating part shown in FIG. It is the perspective view which showed the bag set stand in the measurement part of the nucleic acid detection apparatus shown in FIG. It is the perspective view which showed the state which rotated the bag holding member in the bag set stand shown in FIG. It is the perspective view which showed the chip | tip disposal bag in the measurement part of the nucleic acid detection apparatus shown in FIG. FIG. 22 is an enlarged perspective view of a dripping removal member in the measurement unit of the nucleic acid detection device shown in FIG. 21. FIG. 23 is an enlarged plan view showing a lid closing mechanism of a reaction detection unit in the measurement unit of the nucleic acid detection device shown in FIG. FIG. 23 is a partial side view showing a lid closing mechanism of a reaction detection unit in the measurement unit of the nucleic acid detection device shown in FIG. FIG. 23 is a front view showing a lid closing mechanism of a reaction detection unit in the measurement unit of the nucleic acid detection device shown in FIG. 22. FIG. 23 is a plan view showing a lid closing mechanism part of a reaction detection part in the measurement part of the nucleic acid detection device shown in FIG. 22. FIG. 46 is a right side view of the lid closing mechanism shown in FIG. 45. FIG. 23 is a schematic plan view for explaining a dripping removal operation in a dripping removal member of the measurement unit of the nucleic acid detection device shown in FIG. 22. FIG. 23 is a schematic plan view for explaining a lid pressing operation of a lid closing mechanism in the measurement unit of the nucleic acid detection device shown in FIG. 22.

Explanation of symbols

10 Dispensing mechanism (dispensing means)
11 Arm part 12 Syringe part 12a Nozzle part 12b Pump part 41 Pipette tip (dispensing tip)
60, 460 Reaction detection unit (amplification means, detection means)
60a, 460a Reaction detection block 61 Reaction section (amplification means)
62 Turbidity detector (detection means)
62a LED light source (light source)
62b Photodiode light receiving part (light detecting part)
63,461 lid closing mechanism
6 5 Detection cell (detection container)
66 Cell member 66a Cell part 67 Lid member 67a Lid part (lid)
100 Nucleic acid detector (gene amplification detector)
101, 401 measurement unit 102 data processing unit 461a lid side support member
4 64a Lid pressing member (pressing member)

Claims (12)

A detection container mounting portion provided with a mounting hole for mounting a detection container provided with a lid; and
A lid closing member for placing the lid of the detection container, which is provided so as to be rotatable with respect to the detection container placement section, and is placed in the placement hole;
Dispensing means for dispensing a reagent and a sample suspected of containing a target nucleic acid to the detection container placed in the placement hole ,
A drive unit that drives the lid closing member to close the opening of the detection container with the lid after dispensing of the reagent and the sample into the detection container is completed;
Amplifying means for amplifying a target nucleic acid in a detection container whose lid is closed by the lid closing member ;
A nucleic acid detection apparatus comprising: detection means for detecting the presence of a target nucleic acid in a detection container with the lid closed.   The dispensing means includes a nozzle part to which a dispensing tip is detachably attached to a tip thereof, and a pump part connected to the nozzle part for aspirating and discharging the sample and the reagent. 1. The nucleic acid detection apparatus according to 1.   The nucleic acid detection apparatus according to claim 1, wherein the detection unit includes a light source that irradiates light to the liquid in the detection container, and a light detection unit that detects light emitted from the light source.   The nucleic acid according to any one of claims 1 to 3, wherein the detection means detects the presence of the target nucleic acid by detecting a turbidity of a mixed solution of the sample and the reagent in the detection container. Detection device.   The nucleic acid detection apparatus according to claim 4, wherein the detection unit detects the presence of the target nucleic acid by detecting a change in turbidity of a mixed solution of the sample and the reagent in the detection container.   The nucleic acid detection apparatus according to any one of claims 1 to 3, wherein the detection means detects the presence of the target nucleic acid by detecting a reagent that binds to the amplification product of the target nucleic acid.   The nucleic acid detection apparatus according to claim 1, wherein amplification of the target nucleic acid by the amplification unit and detection of the target nucleic acid by the detection unit are performed in parallel.   The nucleic acid detection apparatus according to claim 1, wherein the target nucleic acid is a cytokeratin nucleic acid.   The nucleic acid detection apparatus according to claim 1, wherein the amplification of the target nucleic acid by the amplification unit is performed by a LAMP method. The nucleic acid detection device according to any one of claims 1 to 9, wherein the reagent and the sample are dispensed into the detection container by the dispensing means in a state where a lid of the detection container is opened. . The lid closing Close the lid by members is performed only once for one of the detection vessel, a nucleic acid detection device according to any one of claims 1 to 10. Further comprising a lid closing member from above the pressing member in addition a pressing force to complete the lid closing operation of the lid by the lid closed part material from above to the lid by pressing downward, claim The nucleic acid detection device according to any one of 1 to 11 .

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