Patent Application
20230304070
The present invention relates to a living body analysis device that analyzes living body substances such as DNA. In particular, the invention relates to a device that performs a pretreatment step for analyzing DNA to be analyzed with a device.
Genomic medicine started fully, and a cancer gene panel diagnostic agent was covered by insurance. A gene profiling test used in cancer gene panel diagnosis is a test in which a nucleic acid is extracted from blood or a pathological specimen collected from a cancer patient, and the nucleic acid is comprehensively analyzed by a next-generation DNA sequencer using a gene mutation test kit reagent.
A treatment policy is determined by a specialist group such as a doctor, a pathologist, a bio-informatician, or a pharmacist based on the analysis result, and then an anticancer agent, a molecular target drug, or an immune checkpoint inhibitor suitable for a patient is provided.
However, since a gene panel test requires a high cost and the number of times that one patient can use the gene panel test in the insurance application is only one, all the tests are required to be successful. Meanwhile, at present, 10 to 20% of specimens are defective as an analysis result.
There are various pretreatment steps for a nucleic acid sample. In the pretreatment step, dispensing of a reagent, amplification of the nucleic acid sample performed by a thermal cycler, purification of DNA, and the like are performed basically by a hand except for some operations. These series of steps have a large number of steps and may have expert knowledge, and there is an increasing need for automation so as not to depend on a skill level of a technician.
A pretreatment automation device that is already commercially available is generally a large device because of units that perform various processes, a gripper for moving a plate between the units, a dispensing machine for dispensing a reagent, and the like.
Meanwhile, in a next-generation sequencer, a large number of DNA fragments to be analyzed, which have been subjected to pretreatment, are fixed on a substrate, and base sequences of the large number of DNA fragments are determined in parallel.
Non-PTL 1 discloses a DNA decoding technique of a next-generation DNA sequencer based on fluorescence detection. In a reaction vessel called a flow cell, clusters in which a plurality of identical DNA fragments are densely packed by an amplification process are disposed at a high density. When a solution containing four types of bases (A, T, G, and C) labeled with four types of phosphors is introduced into the flow cell, complementary bases in the DNA fragments are incorporated by an extension reaction of a polymerase.
Since terminals of the fluorescently labeled bases are modified with a functional group (terminator) for inhibiting the extension reaction, more than one base is not incorporated per DNA fragment. After the extension reaction, excess floating bases are washed away, fluorescence emitted from a reaction spot is detected as a fluorescence spot, and a type of a phosphor is identified by difference in color. After fluorescence detection, terminators and the phosphor on the DNA fragment are dissociated by a chemical reaction to allow a next base to be incorporated.
The extension reaction, fluorescence detection, and terminator dissociation described above are sequentially repeated to decode a sequence of the DNA fragment by about 100 bases.
PTL 1 discloses a method in which a large number of beads on which DNA fragments to be a sample are fixed are disposed in a flow cell, a reagent is supplied to the flow cell, a fluorescence signal generated along with an extension reaction of a base is detected, and a base sequence of the sample is analyzed.
In a pretreatment integrated nucleic acid analysis device, it is necessary to introduce the DNA fragments that have been subjected to a pretreatment step into the flow cell and convey the DNA fragments to an analysis device part that performs a sequence. A work of introduction into the flow cell and conveyance to the device that performs the sequence is currently performed by a hand, and automatic conveyance is also required in accordance with automation of the pretreatment step.
In the pretreatment step, it is necessary to perform various processes such as dispensing and mixing of reaction reagents and a temperature control process such as a PCR reaction. In order to automate the pretreatment step, it is necessary to mount a dispensing machine that dispenses the reagents, a temperature control mechanism that performs the PCR reaction or the like, a conveyance mechanism that conveys a plate or the like containing a sample between the mechanisms, a tip used when dispensing the reagents, and the like.
In general, a robot arm that performs an XYZ drive mechanism and a grip mechanism that holds a plate or the like that is a conveyance object are mounted as a conveyance mechanism mounted in a pretreatment automation device.
A gripper that holds the conveyance object is provided below a Z-axis, and a region that can be held by the gripper and a range within which the conveyance object can be conveyed are determined by a stroke of a drive mechanism that drives X- and Y-axes. Therefore, when widening a region accessible by the gripper, it is necessary to enlarge the drive mechanism for XY driving.
In the pretreatment integrated nucleic acid analysis device, the conveyance mechanism conveys an observation object such as the flow cell to a part where an analysis is performed in addition to a range within which a normal pretreatment step is performed, and thus the range to be accessed by the conveyance mechanism becomes wide.
In the pretreatment integrated nucleic acid analysis device, the drive mechanism for the conveyance mechanism is increased in size, and the entire device is increased in size.
Therefore, there is a demand for a technique for widening the accessible range of the conveyance mechanism while limiting the increase in size of the pretreatment integrated nucleic acid analysis device.
An object of the invention is to implement a pretreatment integrated nucleic acid analysis device capable of widening an accessible range of a conveyance mechanism while limiting an increase in size of the device.
In order to achieve the above object, the invention is configured as follows.
A pretreatment mechanism-integrated nucleic acid analysis device includes: an analysis part configured to analyze a specimen; and a pretreatment part configured to perform pretreatment on the specimen and move the pretreated specimen to the analysis part. The pretreatment part includes a dispensing machine configured to dispense a fluid, a holding mechanism configured to hold a member, and a conveyance mechanism configured to move the holding mechanism in a plane direction, an up-down direction, and a rotation direction.
According to the invention, it is possible to implement a pretreatment integrated nucleic acid analysis device capable of widening an accessible range of a conveyance mechanism while limiting an increase in size of the device.
Problems, configurations, and effects other than those described above will become apparent from the following description of the embodiments.
Hereinafter, an embodiment of the invention will be described in detail with reference to the drawings.
Steps, components, and the like for forming components of the embodiment are not limited to the following description. In addition, when a shape and a function of a component are referred to, a component having a shape or a function similar to the shape and arrangement of the component is included unless otherwise specified. In addition, if a main function is not lost even if a part of the components is changed, the components are regarded as equivalent to those before the change.
A pretreatment integrated nucleic acid analysis device according to the invention can perform, by using a flow cell, various analyses such as determination of a DNA sequence. By setting a gene extracted from a pathological part or the like in a device, it is possible to automatically perform a pretreatment necessary for a DNA sequence, introduce a DNA fragment into the flow cell for observation, and perform the DNA sequence by a sequencer.
In
In addition, the pretreatment mechanism-integrated nucleic acid analysis device 100 includes a pretreatment part 200 (shown in
The gripper unit 110 performs conveyance in the pretreatment part 200. The dispensing unit 106 is driven by a dispensing mechanism actuator 107, and the gripper unit 110 is driven by a gripper actuator 111.
Operations of the analysis part and the pretreatment part 200 described above are controlled by a control PC 104.
The light source unit 101, the stage unit 103, and the optical unit 112 are provided on a vibration isolation table such that a positional relation between the stage unit 103 and the optical unit 112 is maintained even when there is vibration from an outside.
The reagent storage unit 102 includes a cooling part 1000 (to be described later) using a Peltier element 1103 (to be described later), and can mount a plurality of reagent bottles containing a reagent to be used in the sequence. Although not shown, tubes are connected to the reagent bottles mounted in the reagent storage unit 102. The tubes connected to the reagent bottles are connected to the stage unit 103 by a two-way valve, a three-way valve, a rotary valve, or the like.
The stage unit 103 has an injection opening at a position where the flow cell 800 is mounted, and is connected to an inflow hole of the flow cell 800. An outflow hole of the flow cell 800 is connected to a tube connected to a waste liquid tank. A syringe is provided in a middle of the tube connected to the outflow hole of the flow cell 800, and a reagent mounted in the reagent storage unit 102 flows into the flow cell 800 by operating the syringe to perform various reactions necessary for the sequence.
A living body substance such as DNA to be analyzed, the reagent necessary for the pretreatment, and the flow cell 800 are set in the pretreatment mechanism 105. The living body substance set in the pretreatment mechanism 105 is subjected to a process necessary for analysis using the reagent for pretreatment by the pretreatment part 200.
The pretreated living body substance (specimen) is introduced into the flow cell 800 by using the dispensing unit 106 included in the pretreatment part 200. The flow cell 800 is held by a loading unit (holding part) 109, and the specimen is introduced in this state.
Thereafter, the flow cell 800 is mounted on the stage unit 103 of the analysis part by the gripper unit 110 provided in the pretreatment part 200, and the sequence is performed by the analysis part.
In
The gripper 209 is moved in a plane direction by the gripper X-axis actuator 205 and the gripper Y-axis actuator 206, and the gripper 209 is moved in an up-down direction by the gripper Z-axis actuator 207. In addition, the gripper 209 is moved in a rotation direction by the gripper θ-axis actuator 208.
A plurality of dispensing machines 204 are mounted in order to enable simultaneous processing of a plurality of specimens, and dispensing operation can be controlled independently of each other. Each of the dispensing machines 204 may be, for example, a pneumatic electric pipetter driven by a cylinder. Although not shown, a dispensing mechanism necessary for dispensing operations (an aspiration operation and a discharge operation) is incorporated in the dispensing machine 204, and the dispensing mechanism includes a cylinder in which a plunger is incorporated, a motor that reciprocates the plunger, and the like.
In addition, a plurality of mounted dispensing machines 204 are arranged side by side, and are arranged such that the aspiration and discharge operations can be performed in accordance with a plate or a tube having standard dimensions defined by American Standards Association (ANSI) and Biomolecular Chemical Standards Association (SBS).
In addition, each of the dispensing machines 204 includes, for example, a capacitive liquid level detection unit, and feeds back detection of a liquid level to control of the dispensing machine Z-axis actuator 203 during a reagent aspiration operation. The dispensing machine 204 lowers in a Z-axis direction by a specified amount after the liquid level detection, and performs the reagent aspiration operation. The dispensing machine Z-axis actuator 203 is driven in conjunction with the aspiration operation of the dispensing machine 204.
By the operation, it is possible to aspirate a specified amount of reagent even when a reagent amount of an aspiration destination is small. At the time of discharging the reagent, the liquid level detection is performed in the same manner as that at the time of aspirating the reagent, and then a Z-axis is raised or lowered in accordance with the liquid to be discharged, and the discharge operation is performed. The operation prevents air bubbles from being mixed into the discharged solution.
Each of the dispensing machine XYZ-axis actuators 201, 202, and 203 may be, for example, an electric actuator driven by a belt. Although not shown in the drawings, the actuator of each axis includes a motor or the like necessary for driving. In addition, a PI sensor and an encoder that checks a movement accuracy are provided such that an origin of each axis can be detected.
The axes for driving the dispensing machine 204 and the gripper 209 have different origins. For example, as shown in
In the pretreatment mechanism-integrated nucleic acid analysis device 100 according to the present embodiment, a disposable tip is used to prevent contamination between samples. The dispensing machine 204 moves to a position where the tip racks are stored by the dispensing machine X-axis actuator 201 and the dispensing machine Y-axis actuator 202. Thereafter, the dispensing machine Z-axis actuator 203 is driven to be lowered to equip a tip. After the tip is equipped, the dispensing machine Z-axis actuator 203 is driven to be raised to a position of a height that does not interfere with other units, and moves to a reagent aspiration position of a reagent storage part or the like by the dispensing machine X-axis actuator 201 and the dispensing machine Y-axis actuator 202.
The dispensing machine 204 drives in a range indicated by a dispensing machine operation range 210 shown in
In
The gripper 209 is moved to a specified position by the gripper X-axis actuator 205 and the gripper Y-axis actuator 206, and then rotatably moved by the gripper θ-axis actuator 208 in accordance with an orientation of a holding object. Thereafter, the gripper 209 is lowered to a holding position by the gripper Z-axis actuator 207, and holds the holding object.
Similarly at a movement destination, the gripper 209 rotates in accordance with a provision orientation of the holding object. By a planar driving performed by the gripper X-axis actuator 205 and the gripper Y-axis actuator 206 and the rotational driving performed by the gripper θ-axis actuator 208, a conveyance operation can be performed within the range indicated by a gripper operation range 211 shown in
This is because the gripper 209 can be rotationally driven and thus can move to a region beyond the dispensing machine operation range 210.
In the pretreatment mechanism-integrated nucleic acid analysis device 100 according to the present embodiment, the conveyance mechanism conveys the tip rack containing a tip for a dispensing machine, a well plate for mixing reagents, and the flow cell that introduces the pretreated living body substance.
In
The gripper θ-axis actuator 208 includes a θ-axis rotation shaft 208A that rotates the gripper 209 to change an orientation. The grip arm 302 has two arms facing each other. The translation parts 303 are members that drive the two arms of the grip arm 302 simultaneously. An extension line of a straight line connecting the two arms of the grip arm 302 to each other is orthogonal to the Z-axis direction.
The screw mechanism 304 is configured such that an orientation of a screw thread is reversed in a central part, and the translation parts 303 disposed on both sides of the screw mechanism 304 can be driven by one grip arm actuator 301. The grip arm 302 is coupled to the translation parts 303 by the elastic body 305, and is driven in accordance with the translation parts 303.
After the gripper 209 is moved to an upper part of a holding object by the gripper X-axis actuator 205, the gripper Y-axis actuator 206, and the gripper θ-axis actuator 208, the screw mechanism 304 is driven by the grip arm actuator 301. Driving the screw mechanism 304 increases an interval between the translation parts 303. When the translation parts 303 move, the translation parts 303 move in a direction in which an interval between the grip arm 302 increases through the elastic body 305.
After the interval between the grip arm 302 is widened to a specified width, the gripper Z-axis actuator 207 is driven to be lowered to a position where the grip arm 302 reaches a side surface of the holding object. Thereafter, the screw mechanism 304 is reversely rotated by the grip arm actuator 301, and is driven in a direction in which the interval between the two arms of the grip arm 302 is narrowed. The grip arm actuator 301 holds the object by adjusting a driving amount of the screw mechanism 304 in accordance with a width of the object in a holding direction.
After the gripper 209 holds the holding object, the gripper Z-axis actuator 207 is driven to be raised to a position where the gripper Z-axis actuator 207 does not interfere with other units, and moves to a conveyance destination by the gripper X-axis actuator 205, the gripper Y-axis actuator 206, and the gripper θ-axis actuator 208.
In
The reagent mixing unit 502 and the reagent storage unit 504 include a Peltier element 1103 to be described later, a heat sink 1104 for air cooling, an air cooling fan 1101, and a cooling block 1102, and are temperature-controlled to a constant temperature.
The cooling block 1102 is a block in which holes by which a plate or a tube having a standard dimension defined by American Society of Standards (ANSI) and Society of Biomolecular Chemical Standards (SBS) can be provided are formed. Although not shown, the tip rack 503 is disposed on a plane that can be driven in the up-down direction, and a plurality of tips are stacked in a vertical direction.
The reagent for pretreatment is provided on the reagent storage unit 504 and dispensed to a plate provided on the reagent mixing unit 502 by the dispensing machine 204. The plate into which the reagent is dispensed is stirred and mixed by the dispensing machine 204 or a shaker (not shown).
After the stirring, the plate is held by the gripper 209 and conveyed to the thermal cycler 501. Similarly to the reagent mixing unit 502 and the reagent storage unit 504, the thermal cycler 501 also includes a block having a groove conforming to a shape of a bottom surface of the plate.
The thermal cycler 501 includes, for example, a sliding lid driven by a ball screw. After the plate is moved to the thermal cycler 501, a lid for preventing evaporation is placed as necessary, and the lid of the thermal cycler 501 is closed. A heater is incorporated in the lid of the thermal cycler 501, and an upper surface of the plate is brought into a high temperature state while a temperature control reaction such as PCR is performed, thereby preventing a decrease in reaction efficiency and a decrease in the amount of reaction liquid due to evaporation or the like.
The plate generally has a rectangular shape. In addition, since the reagent mixing unit 502 and the thermal cycler 501 mounted on the pretreatment part 200 have a block conforming to the shape of the plate, each component is generally disposed such that the plate is provided in the same direction.
In the present embodiment, since the drive mechanism of the gripper 209 has a rotation shaft, it is not necessary to align plate provision directions of parts at a movement destination of the plate.
As shown in
Thereafter, as shown in
Because of the operation, it is not necessary to align the provision directions of the plates between units that performs conveyance, and it is possible to provide the units in a space-saving manner in accordance with a shape of each part.
The pretreatment mechanism-integrated nucleic acid analysis device 100 according to the invention uses disposable tips. The tips are arranged in eight rows and twelve columns in the tip rack 503, and 96 tips are contained in one tip rack 503. In the pretreatment step, it is necessary to dispense and mix various reagents. Therefore, in order to complete a pretreatment step once, tips corresponding to a plurality of tip racks 503 are required.
In
On a tip rack table 703 of the pretreatment mechanism-integrated nucleic acid analysis device 100, the plurality of tip racks 503 are disposed in a vertically stacked state. The empty tip rack 503 using all the tips is held by the gripper 209. Thereafter, the gripper X-axis actuator 205 and the gripper Y-axis actuator 206 move the gripper 209 to the vicinity of the empty rack placement location 704, and the gripper θ-axis actuator 208 rotates the orientation of the gripper 209 by 180°. After the rotation by 180°, the gripper X-axis actuator 205 and the gripper Y-axis actuator 206 are driven again, and the empty tip rack 503 is moved to the empty rack placement location 704 disposed on a wall side of the pretreatment mechanism-integrated nucleic acid analysis device 100.
Then, a state shown in
In
The flow cell case 805 includes a plurality of positioning holes 801 into which positioning pins are inserted. Meanwhile, the loading unit 109 and the stage unit 103 include the positioning pins corresponding to the positioning holes 801 formed in the flow cell case 805.
In
In the pretreatment mechanism-integrated nucleic acid analysis device 100 according to the present embodiment, it is necessary to accurately align a position of the inflow hole 802 of the flow cell with respect to the injection opening of the loading unit 109 and the provision position of the glass substrate 804 with respect to the stage unit 103.
In
In a case where there is a deviation in the plane direction when the flow cell 800 is provided on the stage unit 103, the flow cell case 805 moves along the taper of the positioning pin 901. In the case of
In
In
As described above, according to the embodiment of the invention, the pretreatment mechanism-integrated nucleic acid analysis device 100 includes the gripper X-axis actuator 205 that moves the gripper 209 that is the conveyance mechanism that conveys the flow cell 800 and the like in an X-axis direction, the gripper Y-axis actuator 206 that moves the gripper 209 in a Y-axis direction, the gripper Z-axis actuator 207 that moves the gripper 209 in the Z-axis direction, and the gripper θ-axis actuator 208 that moves the gripper 209 in a θ-axis direction.
With the above configuration, the gripper 209 is movable not only in the X-axis direction, the Y-axis direction, and the Z-axis direction, but also in the θ-axis direction (rotation direction), and a movement range of the gripper 209 that is the conveyance mechanism can be expanded. The conveyance mechanism (the gripper X-axis actuator 205, the gripper Y-axis actuator 206, the gripper Z-axis actuator 207, the gripper θ-axis actuator 208, and the gripper 209) can be reduced in size while securing a necessary movement range of the flow cell or the like by the pretreatment part 200. That is, it is possible to implement a pretreatment integrated nucleic acid analysis device capable of widening the accessible range of the conveyance mechanism while limiting an increase in size of the device.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/033079 | 9/1/2020 | WO |
