JP4095886B2 - Chemical analysis device and genetic diagnosis device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a chemical analyzer that extracts a specific chemical substance such as nucleic acid from a biological sample such as blood or urine, and mixes the extracted chemical substance such as nucleic acid with a reagent for detection. The present invention also relates to a genetic diagnosis apparatus having the chemical analysis apparatus.
[0002]
[Prior art]
As a chemical analysis apparatus for extracting and analyzing a specific chemical substance such as a nucleic acid from a sample containing a plurality of chemical substances, Japanese Patent Publication No. 2001-527220 discloses an integrated fluid operation cartridge. In this apparatus, a reagent such as a lysing solution, a washing solution, or an eluent, and a capture component for capturing nucleic acid are provided inside the integrated cartridge. After injecting a sample containing nucleic acid into the cartridge, the sample and the eluent are mixed. And pass through the capture component, further pass the cleaning liquid through the capture component, further pass the eluent through the capture component, and contact the eluent after passing through the capture component with the PCR reagent to the reaction chamber. It is flowing.
[0003]
As a nucleic acid extraction method used in the first conventional technique, a method for purifying and separating a nucleic acid mixture by chromatography is described in JP-T-8-501321. In this method, a nucleic acid mixed solution is adsorbed on an inorganic substrate such as silica gel from an adsorption aqueous solution containing a high-concentration salt, then washed with a washing solution, and nucleic acid is eluted with a solution containing a low-concentration salt. Silica gel is fixed in a cylindrical hollow column, and a solution of a nucleic acid mixture to be separated is poured, and the solution is passed through an inorganic substrate by suction or centrifugation.
[0004]
WO 00/78455 describes a microstructure and a method for inspection using amplification. In this apparatus, the DNA mixture is passed through a glass filter as an inorganic substrate using the method for purifying and separating nucleic acid mixture described in JP-A-8-501321. Only recovered. The glass filter is provided in a rotatable structure, and reagents such as cleaning liquid and eluent are held in each reagent reservoir in the same structure. Each reagent flows by centrifugal force generated by the rotation of the structure, and the reagent passes through the glass filter by opening a valve provided in a fine flow path connecting each reagent reservoir and the glass filter.
[0005]
Japanese Patent Application Publication No. 2001-502793 discloses an apparatus and method for chemical analysis. In this device, a chamber, flow path, reservoir, and analysis cell are provided inside the disk-shaped member. After injecting a blood sample into the centrifuge chamber, the blood cells and serum are centrifuged, and the serum-coated beads are coated with a reagent. Only the serum is allowed to flow into the reaction chamber, and then a solution for washing is allowed to flow into the reaction chamber. Further, the elution solution is allowed to flow into the reaction chamber, and the elution solution is moved from the reaction chamber to the analysis cell.
[Patent Document 1]
Special table 2001-527220 gazette
[Patent Document 2]
Japanese National Patent Publication No. 8-501321
[Patent Document 3]
WO00 / 78455
[Patent Document 4]
JP-T-2001-502793
[0006]
[Problems to be solved by the invention]
In the integrated fluid operation cartridge described in JP-T-2001-527220, which is the first prior art, when each reagent is pumped, a valve or the like provided in a fine channel connecting each reagent chamber and the capture component Opening the reagent passes the capture component. Further, of the reagent that has passed through the capture component, the cleaning liquid is switched to the waste liquid chamber, and the eluent is switched to the reaction chamber by a valve or the like provided in the flow path between the capture component and each chamber. When pumping multiple reagents, the reagent remains on the channel wall, especially if there is an obstacle such as a valve. There is a possibility of contamination at the part. In addition, when the cleaning liquid and the eluent that have passed through the capture component are switched by a valve or the like and flow into separate chambers, the cleaning liquid that has flowed to the waste chamber first contaminates the upstream flow path such as the valve that switches to the reaction chamber. Therefore, there is a possibility that the cleaning liquid is mixed into the eluent.
[0007]
In the purification and separation method described in JP-A-8-501321, which is the second prior art, the nucleic acid mixture is poured into a cylindrical hollow column on which silica gel is fixed, and the nucleic acid mixture is applied to the silica gel using centrifugal force. Only the nucleic acid is recovered by passing a plurality of reagents after passing through, but the method of injecting each reagent into the hollow column and the method of recovering the washing solution and the eluent passing through the silica gel are not disclosed.
[0008]
In the structure described in WO 00/78455, which is the third prior art, each reagent flows by the action of centrifugal force by opening a valve provided in a fine channel connecting each reagent reservoir and the glass filter. pass. The valve uses wax or the like that melts when heated, but the reagent that has passed through may remain in the valve part and contaminate the recovered DNA. That is, there is a possibility that the DNA mixed solution or the washing solution remaining in the valve portion flows in the process in which the DNA mixed solution or the washing solution remains in the valve portion and the eluent is passed through the glass filter by centrifugal force.
[0009]
In the apparatus described in Japanese Patent Publication No. 2001-502793, which is the fourth prior art, when separating the serum, the disk-shaped member is rotated (revolved) with respect to the central axis outside the disk-shaped member, and the serum is guided to the reaction chamber. In order to rotate (rotate) with respect to the central axis in the disk-shaped member, a rotation mechanism for each revolution and rotation is required, and the apparatus is complicated. Further, when the cleaning solution and the elution solution are guided to the reaction chamber, the piston is driven in a cylinder provided inside the disk-shaped member, and the apparatus is complicated.
[0010]
An object of the present invention is to provide an inexpensive chemical analyzer for analyzing a specific chemical substance in a liquid sample with high accuracy by solving at least one of the above-mentioned problems. Another object of the present invention is to provide a genetic diagnosis apparatus having these chemical analysis apparatuses.
[0011]
[Means for Solving the Problems]
  In order to achieve the above object, the present invention comprises a structure that is rotatably supported, and holds in the structure a capture unit that captures a specific chemical substance in a sample and a liquid that flows through the capture unit. In a chemical analysis apparatus including a plurality of reagent containers, an eluent holding unit that holds an eluent after elution of the specific chemical substance from the capturing unit and a liquid other than the eluent are discarded from the eluent holding unit An eluent disposal channel for discarding a part of the eluent from the waste solution disposal channel and the eluent holding unit is provided, and a connection part between the eluent holding unit and the eluent disposal channel is provided as the eluent disposal channel. This is provided on the outer peripheral side from the innermost peripheral part.
[0012]
  Further, the present invention includes a structure that is rotatably supported, and the structure includes a specific structure in a sample.In a chemical analysis apparatus including a capture unit that captures a chemical substance and a plurality of reagent containers that hold liquid that flows through the capture unit, an eluent after the specific chemical substance is eluted from the capture unit An eluent holding unit for holding, a detection reagent container for supplying a detection reagent to the eluent holding unit, a detection reagent control unit for controlling the flow of the detection reagent, and a detection reagent for the eluent holding unit are provided. An eluent disposal channel is provided upstream from the detection reagent outlet for supplying to the eluent, and a part of the eluent is discarded from the eluent holder, and a part of the eluent is disposed of the eluent disposal channel. After being discarded, the detection reagent is caused to flow into the eluent holding part.
[0013]
  Furthermore, in the above, it is desirable that the detection reagent control unit is an openable vent and an opening mechanism.
[0014]
  Furthermore, in the above, it is desirable that the detection reagent control unit is a reagent dispenser.
[0015]
  Furthermore, the present invention includes a rotating structure that is rotatably supported, and a plurality of reagents that hold a capturing part that captures a specific chemical substance in a sample and a liquid that flows through the capturing part in the structure. In the chemical analyzer comprising the container, an eluent holding part for holding the eluent after passing through the trapping part and an outflow channel for flowing out the liquid of the eluent holding part are provided, and the eluent holding part and the An outlet channel inlet that is a connection part of the outlet channel is provided on the inner peripheral side from the outlet channel outlet that is the other end of the outlet channel, and the reagent that has flowed through the trapping part during rotation of the rotating structure is After flowing through the outflow channel, the rotating structure is once stopped and then rotated again, and after being stopped again, the eluent is allowed to flow through the capturing part.
[0022]
DETAILED DESCRIPTION OF THE INVENTION
Example 1
An embodiment of a chemical analyzer according to the present invention will be described with reference to FIGS.
[0023]
FIG. 1 is an overall configuration diagram of a gene analyzer according to the present invention. The gene analyzer 1 includes a holding disk 12 rotatably supported by a motor 11, a plurality of fan-shaped analysis disks 2 positioned by protrusions 121 on the holding disk 12, and a punch for controlling the flow of liquid. 13 and two optical devices for heating and detection, that is, an upper optical device 14 and a lower optical device 15, and a positioning sensor 16 to be described later (FIG. 18). The holding disk 12 includes a holding disk optical window 122 for the lower optical device 15.
[0024]
FIG. 2 is a configuration diagram of the analysis disk 2. The analysis disk 2 is configured by joining the upper cover 20 and the flow path portion 30. The upper cover 20 includes a sample inlet 210, a plurality of reagent inlets 220, 230, 240, 250, 260, 270, a plurality of vent holes 212, 222, 223, and a plurality of lidded vent holes 221, 231, 241. , 251, 261, 271, 272, 273, 274. The flow path unit 30 includes a positioning hole 710 and a container and a flow path described later. The analysis disk 2 is positioned by fitting the positioning hole 710 with the protrusion 121 of the holding disk 12.
[0025]
The block diagram of the flow path part 30 is shown in FIG. In the embodiment of the flow path section shown in FIG. 3, after separating serum from whole blood, the nucleic acid contained in the virus in the serum is extracted, and the extract from which the nucleic acid has been extracted is quantified and added with a detection reagent for analysis. Constitutes the road.
[0026]
The viral nucleic acid extraction and analysis operations when whole blood is used as a sample will be described below. The flow of extraction and analysis operation is shown in FIGS. 4 and 5, and the flow state in the flow path section 30 is shown step by step in FIGS.
[0027]
The operator dispenses the reagent from the upper cover 20 of the analysis disk 2 to the reagent containers 320, 330, 340, 350, 360, 370 through the reagent inlets 220, 230, 240, 250, 260, 270, and covers the lids. To do. After injecting the reagent into the required number of analysis disks according to the number of analysis, the analysis disk is mounted on the holding disk 12.
[0028]
Next, whole blood collected by a vacuum blood collection tube or the like is injected into the sample container 310 from the sample injection port 210 (FIG. 6).
After injecting whole blood 501, the holding disk 12 is rotated by the motor 11. The whole blood injected into the sample container 310 flows to the outer peripheral side by the action of the centrifugal force generated by the rotation of the holding disk 12, fills the blood cell storage container 311 and the serum quantitative container 312, and excess whole blood flows into the overflow tubule flow. It flows from the path 313 to the whole blood disposal container 315 through the overflow thick pipe channel 314 (FIG. 7). The whole blood disposal container 315 is provided with a whole blood disposal aeration channel 318, and a whole blood disposal vent 212 is provided at a position corresponding to the innermost periphery of the whole blood disposal aeration channel 318 of the upper cover 20. Since it is provided, air can freely enter and exit. Since the connection from the overflow narrow tube flow path 313 to the overflow large tube flow path 314 is abruptly expanded and is located on the innermost peripheral side (radius position 601) of the overflow thin tube flow path 313, whole blood flows into the overflow narrow tube flow path 313. It cuts at the connecting portion in a state where Accordingly, since no liquid can exist on the inner peripheral side from the radial position 601, the liquid level of the serum quantitative container 312 also becomes the radial position 601. The whole blood also flows into the serum capillary 316 branched from the serum quantification container 312, and the innermost peripheral portion of the whole blood is at the radial position 601 here.
[0029]
When the rotation is further continued, the whole blood 501 is separated into blood cells and serum (centrifugation), the blood cells 502 move to the blood cell storage container 311 on the outer peripheral side, and only the serum 503 is contained in the serum quantitative container (FIG. 8).
[0030]
During the above series of serum separation operations, the vent holes 221, 231, 241, 251 and 261, 271 of each reagent container in the upper cover 20 are covered so that air does not enter. Each reagent tries to flow out from the outer peripheral side of the reagent container due to the centrifugal force. However, since air does not enter the container, the pressure in the reagent container decreases, and the reagent cannot flow out in balance with the centrifugal force. However, when the rotational speed increases and the centrifugal force increases, the pressure in the reagent container gradually decreases, and bubbles are generated when the pressure is lower than the saturated vapor pressure of the reagent. Therefore, as shown in FIG. 6, a flow path structure (return flow paths 322, 332, 342, 352, 362, 372) that once returns the reagent flowing out from the outer peripheral side of each reagent container to the inner peripheral side is adopted. , Suppresses the pressure drop in the reagent container and prevents the generation of bubbles. Thus, during the serum separation operation, each reagent does not flow while being held in the reagent container.
[0031]
When the serum separation operation is completed by rotating for a predetermined time, the analysis disk 2 is stopped, a part of the serum 503 in the serum quantitative container 312 is capillary-flowed inside the serum capillary 316 due to surface tension, and the mixing unit 410 and the serum capillary 316 To the mixing part inlet 411, which fills the serum capillary 316.
[0032]
Thereafter, the perforator 13 opens holes one by one in the lid of the vent hole at the top of each reagent container, and the motor 11 is rotated to cause each reagent to flow by centrifugal force. As shown in the analysis disk cross section of FIG. 18A, the upper cover 20 has reagent inlets (240, 250, 260) and vent holes (241, 251 and 261) at the upper part of each reagent container. Has a lid. The perforator 13 makes a state in which air enters the reagent container by making a hole in the lid. Furthermore, as shown in FIG. 18 (b), contamination of the perforator 13 can be prevented by providing filters 242, 252, and 262 between the vent hole and the reagent container.
[0033]
The operation after serum separation is shown below.
In the lysing solution container 320, a lysing solution 521 for dissolving a viral membrane protein in serum is dispensed. When the motor 11 is rotated after the perforator 13 has made a hole in the lid of the solution vent hole 221, the solution 521 passes from the solution container 320 through the solution return flow path 322 by the action of centrifugal force, and the mixing unit. Flow into 410. Further, since the innermost peripheral side of serum in the serum quantitative container 312 (radius position 601 at the end of serum separation) is located on the inner peripheral side from the mixing unit inlet 411 (radial position 602), the serum quantitative container is caused by a head difference due to centrifugal force. The serum in 312 and the serum capillary 316 flows into the mixing unit 410 from the mixing unit inlet 411 (FIG. 9). The mixing unit 410 is composed of a member that mixes serum and lysate. For example, porous filters and fibers such as resin, glass and paper, or protrusions such as silicon and metal manufactured by etching or machining.
[0034]
Serum and lysate are mixed in the mixing section 410 and flow into the reaction container 420 (FIG. 10). The reaction vessel 420 is provided with a reaction vessel vent channel 423, and the reaction vessel vent hole 222 is provided at a position corresponding to the innermost peripheral portion of the reaction vessel vent channel 423 of the upper cover 20. Air can enter and exit freely. Since the branching portion 317 (radius position 603) from the serum quantitative container 312 to the serum capillary 316 is located on the inner peripheral side from the mixing portion entrance 411 (radius position 602), all the serum in the serum capillary 316 is mixed by the siphon effect. Flows out. On the other hand, since the serum in the serum quantitative container 312 flows into the serum capillary 316 by centrifugal force, the serum flows out to the mixing part 410 until the liquid level of the serum in the serum quantitative container 312 reaches the branching portion 317 (radius position 603). Subsequently, when the serum level reaches the branching portion 317, air enters the serum capillary 316 and becomes empty, and the flow ends. That is, the serum in the serum quantitative container 312, the overflow capillary channel 313 and the serum capillary channel 316 from the radial position 601 to the radial position 603 at the end of serum separation flows out into the mixing unit 410 and is mixed with the lysate.
[0035]
As described above, if the serum quantification container 312, the overflow capillary channel 313 and the serum capillary channel 316 from the radial position 601 to the radial position 603 are designed to have a predetermined volume (required serum volume), Even if the ratio is different for each whole blood sample, the serum used for the analysis can be quantified. For example, when the blood cell storage container has a volume of 250 microliters and the required serum volume is designed to be 200 microliters, if 500 microliters of whole blood is dispensed, 50 microliters of whole blood overflows into the whole blood disposal container 315. The remaining 450 microliters is separated into serum and blood cells, and 200 microliters of the separated serum flows out to the mixing unit 410. That is, with respect to 450 microliters of whole blood, a whole blood sample having a serum amount of 200 microliters or more can be analyzed with the device of the present invention. For whole blood with a low serum ratio, the volume of the blood cell storage container may be increased to increase the number of whole blood samples.
[0036]
In the reaction container 420, the mixed serum and the lysate react. Since the liquid level in the reaction container 420 after the mixed solution of serum and lysate flows into the reaction container 420 is located on the outer peripheral side of the innermost peripheral part (radius position 604) of the reaction liquid channel 421, the reaction liquid The innermost peripheral portion of the flow path cannot be exceeded, and the mixed liquid is held in the reaction vessel 420 during rotation.
[0037]
The lysing solution functions to dissolve the membrane from viruses, bacteria, and the like in the serum to elute the nucleic acid, and further promotes the adsorption of the nucleic acid to the nucleic acid binding member 301 serving as the capturing unit in the present invention. As such a reagent, guanidine hydrochloride may be used for elution and adsorption of DNA, guanidine thiocyanate may be used for RNA, and a porous material such as quartz or glass or a fiber filter may be used as the nucleic acid binding member.
[0038]
After the serum and the lysate are held in the reaction container 420, the motor 11 is stopped, a hole is made in the lid of the additional liquid vent hole 231 for supplying air to the additional liquid container 330 by the punching machine 13, and the motor 11 is again formed. , The additional liquid 531 flows from the additional liquid container 330 through the additional liquid return channel 332 to the reaction container 420 by the action of centrifugal force, and moves the liquid level of the mixed liquid in the reaction container to the inner peripheral side. (FIG. 11). When the liquid level reaches the innermost peripheral part (radius position 604) of the reaction liquid channel 421, the mixed liquid flows out beyond the innermost peripheral part of the reaction liquid channel, passes through the converging channel 422, and the nucleic acid binding member 301. Flow into. As the additional liquid, for example, the above-described dissolving liquid may be used.
[0039]
Depending on the sample, the wettability of the mixed liquid to the wall surface is good, and in the stopped state, the mixed liquid may flow in the reaction liquid channel 421 by capillary action. In this case, the additional liquid 531 is not required.
[0040]
Thus, when the mixed solution of lysate and serum passes through the nucleic acid binding member, the nucleic acid is adsorbed to the nucleic acid binding member, and the liquid flows into the waste liquid storage container 430 through the waste liquid channel 431. There are a plurality of containers and channels on the downstream side of the eluent channel 451, and holes for supplying air to each container are drilled in a later step, but when the mixed solution passes through the nucleic acid binding member 301. Is sealed, and the liquid does not flow into the eluent channel 451. On the other hand, the waste liquid storage container 430 communicates with the pressure adjustment container 440 through the pressure adjustment flow path 432. The pressure adjusting container 440 is provided with a pressure adjusting vent channel 441, and further, the pressure adjusting vent hole 223 is provided at a position corresponding to the innermost peripheral part of the pressure adjusting vent channel 441 of the upper cover 20. Air can enter and exit freely.
[0041]
Next, when the motor 11 is stopped and the hole of the first cleaning liquid vent hole 241 for supplying air to the first cleaning liquid container 340 is opened by the punching machine 13, the motor 11 is rotated again. As a result, the first cleaning liquid 541 flows into the nucleic acid binding member 301 from the first cleaning liquid container 340 via the first cleaning liquid return channel 342, and cleans unnecessary components such as proteins attached to the nucleic acid binding member 301 (FIG. 12). As the first cleaning liquid, for example, the above-described dissolution liquid or a liquid with a reduced salt concentration may be used.
[0042]
The waste liquid after washing flows into the waste liquid storage container 430 through the waste liquid flow path 431 as in the above-described mixed liquid.
[0043]
The same cleaning operation is repeated several times. For example, following the first cleaning liquid, with the motor stopped, a hole is made in the lid of the second cleaning liquid vent hole 241 for supplying air to the second cleaning liquid container 350 by the perforator 13 and the motor 11 is rotated again to bind the nucleic acid. Unnecessary components such as salt adhering to the member 301 are washed. As the second cleaning liquid, for example, ethanol or an aqueous ethanol solution may be used.
[0044]
You may repeat the same washing | cleaning further as needed.
[0045]
In such a cleaning process, each cleaning liquid flows into the waste liquid storage container 430 through the waste liquid flow path 431, but there is a possibility that a part of the eluent flow path 451, in particular, the vicinity of the branch portion with the waste liquid flow path may be contaminated. is there. As will be described later, since the nucleic acid eluted from the nucleic acid binding member 301 passes through the eluent channel 451, it is desirable to wash the eluent channel 451 as well.
[0046]
In Embodiment 1 of FIGS. 6 to 17, a case is described in which cleaning is performed with two types of cleaning liquids, that is, the first cleaning liquid 541 and the second cleaning liquid 551, and the eluent flow path 451 is cleaned with the second cleaning liquid.
[0047]
First, FIG. 13 shows a state where all the first cleaning liquid has passed through the waste liquid flow path 451. The waste liquid overflows from the waste liquid storage container 430 to the pressure adjustment flow path 440 through the pressure adjustment flow path 432. Once the motor is stopped, a hole is made in the lid of the second cleaning liquid vent 241 for supplying air to the second cleaning liquid container 350 by the punching machine 13, and the detection container vent hole for communicating the detection container 450 with the outside. A hole is made in the lid of the final cleaning liquid vent 273 for communicating the 272 and the final cleaning liquid waste container 460 with the outside.
[0048]
When the motor 11 is rotated again, the second cleaning liquid 551 flows from the second cleaning liquid container 350 into the nucleic acid binding member 301 through the second cleaning liquid return channel 352 by the action of centrifugal force, and adheres to the nucleic acid binding member 301. The cleaning solution is cleaned (FIG. 14). The second washing liquid that has passed through the nucleic acid binding member 301 tends to flow into both the detection container 450 and the waste liquid storage container 430, but the waste liquid storage container 430 is used when the liquid flows into the pressure adjustment channel 432. In addition to the head difference (h1), a head difference (h2) for pushing out air from the pressure adjusting flow path 432 to the pressure adjusting container 440 is required, and the second cleaning liquid cannot flow in. On the other hand, the detection container 450 has a vent hole formed by the above-described drilling operation, and therefore flows almost without resistance. That is, the second washing liquid that has passed through the nucleic acid binding member 301 flows into the detection container 450, which is an eluent holding part in the present invention, through the eluent flow path 451. At this time, the vicinity of the branch portion with the waste liquid flow path 431 that is contaminated with the mixed liquid or the first cleaning liquid is cleaned.
[0049]
When the second cleaning liquid flows into the detection container 450 and the liquid level reaches the innermost peripheral portion (radius position 605) of the cleaning liquid disposal flow path 452, it flows out to the final cleaning liquid disposal container 460. Since the connection portion (radius position 606) of the cleaning liquid disposal flow path 452 with the detection container is located on the outer peripheral side from the innermost peripheral portion (radius position 605), once it flows out to the final cleaning liquid disposal container 460, the detection container 450 is caused by the siphon effect. Try to drain all the liquid inside. However, when a small amount of liquid remaining in the nucleic acid binding member 301 or the like flows after the discharge is completed, the liquid remains in the detection container 450. In this case, once the rotation is stopped and the liquid remaining in the detection container 450 fills the cleaning liquid disposal flow path 452 by capillary flow and then rotates again, the liquid remaining in the detection container 450 is again returned to the final cleaning liquid by the siphon effect. Discharge to waste container 460. Therefore, with respect to the final cleaning liquid, it is desirable to repeat the rotation and stop twice after drilling the vent hole.
[0050]
Thus, after the nucleic acid binding member 301 is washed and only the nucleic acid is adsorbed, the process proceeds to the nucleic acid elution step.
[0051]
That is, when the motor is stopped, a hole is made in the lid of the eluent vent hole 261 for supplying air to the eluent container 360 by the punching machine 13, and the eluent disposal container 470 which is the eluent disposal section referred to in the present invention. Is made in the lid of the eluent disposal vent 274 for communicating with the outside. The motor 11 is rotated again to flow the eluent through the nucleic acid binding member 301 (FIG. 15). The eluent is a liquid that elutes nucleic acid from the nucleic acid binding member 301, and water or an aqueous solution whose pH is adjusted from 7 to 9 may be used. In particular, it is desirable to heat to 40 degrees or more in order to facilitate elution. The upper optical device 14 of FIG. 1 may be used for heating, and light may be irradiated from above the eluent container 360.
[0052]
After passing through the nucleic acid binding member 301, the eluent flows into the detection container 450 through the eluent channel 451. Since the eluent disposal container 470 communicates with the outside by the above-described perforating operation, the eluent flows out to the eluent disposal container 470 through the eluent disposal channel 471. Since the connection part (radius position 607) of the eluent disposal channel 471 to the detection container 450 is located on the inner peripheral side from the connection part (radius position 608) to the eluent disposal container 470, the inside of the detection container 450 is caused by the siphon effect. The eluent at the inner periphery from the radial position 607 is discharged to the eluent disposal container 470. In this way, the eluent containing the nucleic acid can be quantified and held in the detection container 450 (FIG. 16).
[0053]
Next, with the motor stopped, a hole is made in the lid of the detection liquid vent hole 271 for supplying air to the detection liquid storage container 370 by the punching machine 13, the motor 11 is rotated again, and the detection liquid 571 is put into the detection container 450. Flow (FIG. 17). The detection solution is a reagent for amplifying and detecting nucleic acid, and includes deoxynucleoside triphosphate, DNA synthase, fluorescent reagent, and the like. Depending on the amplification method, heating may be performed by irradiating light from above the detection container 450 using the upper optical device 14.
[0054]
Next, the lower optical device 15 is moved below the detection container 450 to detect, for example, the amount of fluorescence emitted.
[0055]
It is necessary to stop the holding disk 12 at a predetermined position during the drilling, heating, and detection. As shown in FIG. 19, the holding disk 12 is provided with positioning protrusions 17, the position detector 16 detects the rotation position of the holding disk, and the controller 18 rotates the motor 11 and the drilling machine 13. Motion, rotation, irradiation and detection of the upper optical device 14 and the lower optical device 15 are controlled.
[0056]
For example, FIG. 20 shows the operation timing of the punching machine 13. The holding disk 12 reduces the rotation speed after the flow of whole blood or each reagent is completed, and maintains a low-speed rotation for positioning. When the position detector 16 detects the positioning protrusion 17, the holding disk 12 is stopped, the perforator 13 is lowered, and the vent cover of each reagent storage container is made a hole and then raised again. The perforated holding disk 12 rotates at a low speed so that the reagent does not flow out from the reagent storage container after the end of the perforation, and the position of the next analysis disk, that is, 60 degrees of rotation when six analysis disks are mounted. Stop and repeat the same drilling operation. Where the analysis disk is mounted may be determined by, for example, irradiating light from the channel optical window 490 with the lower optical device and examining the reflected light. After all analysis discs have been drilled, the holding disc rotates at high speed to allow the reagent to flow.
[0057]
According to the present embodiment, there is no need to provide a valve for controlling the flow of the sample and each reagent in the middle of the flow path, and no liquid residue is generated in the valve portion in the middle of the flow path, and the reagent in the previous step Contamination can be prevented, and specific components such as nucleic acids in a liquid sample can be extracted with high purity and analyzed with high accuracy.
(Example 2)
In Example 1 above, serum was separated from whole blood, and nucleic acids in pathogens such as viruses and bacteria contained in the separated serum were extracted and analyzed. However, nucleic acids in white blood cells were extracted and analyzed from whole blood. May be.
With reference to FIGS. 21 to 28, an embodiment of a gene analyzer for extracting and analyzing leukocyte nucleic acids from whole blood will be described.
The overall configuration diagram of the gene analyzer of the present invention is the same as that of FIG. 1, in order to extract and analyze nucleic acids in leukocytes instead of the analysis disk 2 for extracting and analyzing nucleic acids in pathogens such as viruses and bacteria. The white blood cell analysis disk 3 is used.
FIG. 21 is a block diagram of a white blood cell analysis disk. The leukocyte analysis disk 3 is configured by joining the upper cover 90 and the flow path portion 60. The upper cover 90 includes a sample injection port 910, a plurality of reagent injection ports 920, 930, 940, 950, 960, 970, 980, a plurality of vent holes 922, 923, 982, and a plurality of vent holes with lids 921, 931. , 941, 951, 961, 971, 972, 973, 974, 981. The flow path unit 60 includes a positioning hole 720, a container, a flow path, and the like described later. The leukocyte analysis disk 3 is positioned by fitting the positioning hole 720 to the protrusion 121 of the holding disk 12 shown in FIG.
[0058]
A configuration diagram of the flow path section 60 is shown in FIG. The embodiment of the flow path section shown in FIG. 22 constitutes a flow path for extracting nucleic acid contained in white blood cells from whole blood and adding the detection reagent after quantification of the extract.
[0059]
The nucleic acid extraction and analysis operations when whole blood is used as a sample will be described below. The flow of the extraction and analysis operation is shown in FIGS. 23 and 24, and the flow state in the flow path section 60 is shown step by step in FIGS.
[0060]
The operator dispenses the reagent from the upper cover 90 of the analysis disk 3 to the reagent containers 620, 630, 640, 650, 660, 670, 680 through the reagent inlets 920, 930, 940, 950, 960, 970, 980. Put the lid on. After injecting the reagent into the required number of analysis disks according to the number of analysis, the analysis disk is mounted on the holding disk 12.
[0061]
Next, whole blood collected by a vacuum blood collection tube or the like is injected into the sample container 610 from the sample injection port 910 (FIG. 25).
After injecting whole blood 501, the holding disk 12 is rotated by the motor 11. The whole blood injected into the sample container 610 flows to the outer peripheral side by the action of centrifugal force generated by the rotation of the holding disk 12, flows to the lysis container 880, and mixes with the lysis solution 571 in the lysis container 880. Lysates the white blood cells (FIG. 26). The dissolution container 880 is provided with a dissolution container ventilation channel 881, and the dissolution container ventilation hole 982 is provided at a position corresponding to the innermost peripheral portion of the dissolution container ventilation channel 881 of the upper cover 90. It is possible to go in and out.
A proteolytic enzyme such as protease K may be used as the solution.
[0062]
At the time of dissolution, the vent holes 921, 931, 941, 951, 961, 971, 981 of each reagent container in the upper cover 90 are covered so that air does not enter as in the first embodiment. 6 is provided with a return flow path that once returns the reagent flowing out from the outer peripheral side of each reagent container to the inner peripheral side, thereby suppressing the pressure drop in the reagent container and preventing the generation of bubbles.
[0063]
When the lysis of the white blood cells is completed, the analysis disk 3 stops at a predetermined position.
[0064]
Thereafter, the perforator 13 opens holes one by one in the lid of the vent hole at the top of each reagent container, and the motor 11 is rotated to cause each reagent to flow by centrifugal force. The cross section of each reagent container has the structure shown in FIG. 18A or FIG. 18B as in the first embodiment. By making a hole in the lid of the vent hole with the punching machine 13, air is allowed to enter the reagent container. ing.
[0065]
The operation after dissolution is shown below.
If the motor 11 is rotated after the perforator 13 has made holes in the lids of the binding liquid vent hole 921 and the mixed additional liquid vent hole 981, the binding liquid 521 and the mixed additional liquid 581 are combined with the binding liquid container 620 by the action of centrifugal force. The mixed additional liquid 581 flows into the lysis container 880 and the mixed liquid of the whole blood 501 and the lysis reagent 571 (dissolved mixed liquid 572) is expelled from the lysis container 880 to the mixing unit 810, and the binding liquid. 521 and the dissolved mixed solution 572 are mixed in the mixing unit 810 (FIG. 27).
The mixing unit 810 is composed of a member that mixes the dissolved mixed solution and the binding solution. For example, porous filters and fibers such as resin, glass and paper, or protrusions such as silicon and metal manufactured by etching or machining.
The dissolved mixed solution 572 and the binding solution 521 are mixed in the mixing unit 810 and flow into the reaction vessel 820 (FIG. 28). The reaction vessel 820 is provided with a reaction vessel vent channel 823, and further provided with a reaction vessel vent 922 at a position corresponding to the innermost peripheral portion of the reaction vessel vent channel 823 of the upper cover 90. Air can enter and exit freely.
[0066]
In the reaction vessel 820, the dissolved mixed solution and the binding solution react. Since the liquid level in the reaction vessel 820 after the dissolved mixed solution and the binding solution flow into the reaction vessel 820 is located on the outer peripheral side of the innermost peripheral portion (radial position 604) of the reaction liquid channel 821, the reaction liquid flow The innermost peripheral portion of the path cannot be exceeded, and the mixed liquid is held in the reaction vessel 820 during the rotation.
[0067]
The binding liquid promotes the adsorption of the nucleic acid to the nucleic acid binding member 801 which is the capturing unit referred to in the present invention. As such a reagent, guanidine hydrochloride or guanidine thiocyanate may be used, and as a nucleic acid binding member, a porous material such as quartz or glass or a fiber filter may be used. The mixed additional liquid is a liquid for driving out the dissolved mixed liquid, and the mixed liquid 521 and the dissolved liquid 571 are preferable.
[0068]
After the mixed additional liquid and the binding liquid are held in the reaction vessel 820, the same procedure as in the first embodiment is performed. That is, refer to the first embodiment, that is, FIGS. 11 to 16 for the flow state of the liquid, and refer to FIG. 25 for the reference numerals. Note that the same liquid as in the first embodiment may be used as the cleaning liquid and the eluent.
That is, after the mixing shown in FIG. 23, the motor 11 is first stopped, a hole is made in the lid of the additional liquid vent hole 931 for supplying air to the combined additional liquid container 630 by the punching machine 13, and the motor 11 is rotated again. Then, the reaction solution in the reaction vessel 820 is expelled with the additional solution and passed through the nucleic acid binding member. The nucleic acid is adsorbed on the nucleic acid binding member 801, and the liquid flows into the storage container 830.
[0069]
Next, when the motor 11 is stopped and a hole is made in the lid of the first cleaning liquid vent 941 for supplying air to the first cleaning liquid container 640 by the punching machine 13, the motor 11 is rotated again. The liquid in the container flows into the nucleic acid binding member 801, and unnecessary components such as proteins attached to the nucleic acid binding member 801 are washed. The waste liquid after washing flows into the waste liquid storage container 830.
[0070]
After the motor is stopped, a hole is formed in the lid of the second cleaning liquid vent 941 for supplying air to the second cleaning liquid container 650 by the punching machine 13, and the detection container vent hole 972 for communicating the detection container 850 with the outside. A hole is made in the lid of the final cleaning liquid vent hole 973 for communicating the final cleaning liquid disposal container 860 with the outside.
[0071]
When the motor 11 is rotated again, the second cleaning liquid in the second cleaning container 650 cleans the first cleaning liquid attached to the nucleic acid binding member 801. The second washing liquid that has passed through the nucleic acid binding member 801 attempts to flow into both the detection container 850 and the waste liquid storage container 830, but flows into the waste liquid storage container 830 due to the head difference described in the first embodiment. However, it flows into the detection container 850 which is an eluent holding part as referred to in the present invention while washing the branch part of the waste liquid storage container and the detection container.
[0072]
As the second cleaning liquid, for example, ethanol or an aqueous ethanol solution may be used.
[0073]
When the amount of cleaning liquid in the detection container 850 increases, it overflows into the final cleaning liquid disposal container 860, and all the liquid in the detection container 850 is discharged to the final cleaning liquid disposal container 860 by the siphon effect. However, when a small amount of liquid remaining in the nucleic acid binding member 801 or the like flows after the discharge is completed, the liquid remains in the detection container 850. In this case, if the rotation is stopped once and then rotated again after a while, the liquid remaining in the detection container 850 can be discharged to the final cleaning liquid disposal container 860 by capillary action and siphon effect. Therefore, with respect to the final cleaning liquid, it is desirable to repeat the rotation and stop twice after drilling the vent hole.
Next, a hole is made in the lid of the eluent vent hole 961 for supplying air to the eluent container 660 by the punching machine 13, and the eluent waste container 870 which is an eluent waste section referred to in the present invention is communicated with the outside. A hole is made in the lid of the eluent disposal vent hole 974. The motor 11 is rotated again, and the eluent flows through the nucleic acid binding member 801. The eluent may be water or an aqueous solution whose pH is adjusted to 7-9. In particular, it is desirable to heat to 40 degrees or more in order to facilitate elution. The upper optical device 14 of FIG. 1 may be used for heating, and light may be irradiated from above the eluent container 660.
[0074]
After passing through the nucleic acid binding member 801, the eluent flows into the detection container 850 and further flows out to the eluent disposal container 870. At this time, a predetermined amount of eluent containing nucleic acid is held in the detection container 850 as in the first embodiment.
[0075]
Next, with the motor stopped, a hole is made in the lid of the detection liquid vent hole 971 for supplying air to the detection liquid storage container 670 with the punch 13, the motor 11 is rotated again, and the detection liquid is allowed to flow through the detection container 850. . The detection liquid contains deoxynucleoside triphosphate, DNA synthase, fluorescent reagent, and the like. Depending on the detection method, heating may be performed by irradiating light from above the detection container 850 using the upper optical device 14.
[0076]
Next, the lower optical device 15 is moved below the detection container 850 to detect, for example, the amount of fluorescent light emission.
[0077]
It is necessary to stop the holding disk 12 at a predetermined position during the drilling, heating, and detection. As in the first embodiment, as shown in FIGS. 19 and 20, the position detector 16 detects the rotational position of the holding disk, the controller 18 rotates the motor 11, the punch 13 rotates and moves up and down, and the upper optics. The rotation, irradiation, and detection of the device 14 and the lower optical device 15 are controlled.
[0078]
According to the present embodiment, there is no need to provide a valve for controlling the flow of the sample and each reagent in the middle of the flow path, and no liquid residue is generated in the valve portion in the middle of the flow path, and the reagent in the previous step Contamination can be prevented, and specific components such as nucleic acids in a liquid sample can be extracted with high purity and analyzed with high accuracy.
(Example 3)
In Example 1 and Example 2 above, nucleic acids in pathogens such as viruses and bacteria and nucleic acids in white blood cells were extracted and analyzed from whole blood alone, but both may be performed simultaneously.
[0079]
FIG. 29 shows a flow of operations for simultaneously extracting and analyzing pathogen nucleic acids such as viruses and bacteria from whole blood and extracting and analyzing nucleic acids in leukocytes. The flow state at that time is shown in FIG. 30 and FIG.
[0080]
As shown in the flow path part 61 of FIG. 30, the nucleic acid in the pathogen described in the first embodiment is extracted and analyzed, and the nucleic acid in the white blood cell described in the second embodiment is extracted and analyzed. The flow path is configured on a single device.
[0081]
The extraction and analysis operations may be performed simultaneously in the above two embodiments. That is, the operator injects each reagent into the analysis disk, attaches it to the holding disk, and injects whole blood into the sample container 310 (FIG. 30). When the holding disk is rotated by the motor, the whole blood injected into the sample container 310 moves to the outer peripheral side, fills the blood cell storage container 311 and the serum quantification container 312, and excess whole blood flows from the overflow capillary tube 313 to the overflow large tube. It flows through the flow path 314 to the lysis container 880 and is mixed with the lysis solution 571 in the lysis container 880 to lyse leukocytes in the whole blood (FIG. 31).
[0082]
The subsequent operation is the same as that of the first embodiment for the whole blood in the blood cell storage container 311 and the serum quantitative container 312. After the serum separation, the nucleic acid in the pathogen is adsorbed to the nucleic acid binding member 301, and a plurality of washings are performed. After the step, the nucleic acid is eluted from the nucleic acid binding member 301 and finally the nucleic acid is detected by the detection container 450. Similarly, the lysis mixture 572 in which the whole blood and the lysis solution are mixed in the lysis vessel 880 is the same as in the second embodiment, and the nucleic acid in the white blood cells is adsorbed to the nucleic acid binding member 801, and after the plurality of washing steps, The nucleic acid is eluted from the nucleic acid binding member 801, and finally the nucleic acid is detected by the detection container 850.
[0083]
According to this embodiment, since the nucleic acid of the pathogen and the nucleic acid in the white blood cell can be extracted and analyzed from the same whole blood sample, the presence or absence of infection by the pathogen can be confirmed, and at the same time, the administration of the drug from the genome information of the patient The effect can be predicted and the optimal drug can be selected. In particular, since a very small amount of whole blood that is surplus at the time of pathogen nucleic acid extraction is used for predicting the drug administration effect, the burden on the patient at the time of blood collection is small.
Example 4
In Examples 1 to 3, the vent flow holes were opened by the punching machine to control the flow of the reagent, but a reagent dispensing mechanism may be used. That is, as shown in FIG. 32, the reagent dispenser 19 dispenses a predetermined reagent from each reagent bottle 400 into the reagent storage container shown in FIG. 6, FIG. 25 or FIG. Let The procedure is shown in FIG.
[0084]
According to the present embodiment, there is no need to provide a valve for controlling the flow of the sample and each reagent in the middle of the flow path, and no liquid residue is generated in the valve portion in the middle of the flow path, and the reagent in the previous step Contamination can be prevented, and specific components such as nucleic acids in a liquid sample can be extracted with high purity and analyzed with high accuracy.
[0085]
Alternatively, according to the present embodiment, since the nucleic acid of the pathogen and the nucleic acid in the leukocyte can be extracted and analyzed from the same whole blood sample, it is possible to confirm the presence or absence of infection by the pathogen, and at the same time, from the patient's genome information, The administration effect can be predicted and the optimal drug can be selected. In particular, since a very small amount of whole blood that is surplus at the time of pathogen nucleic acid extraction is used for predicting the drug administration effect, the burden on the patient at the time of blood collection is small.
[0086]
【The invention's effect】
According to the present invention, it is not necessary to provide a valve for controlling the flow of the sample and each reagent in the middle of the flow path, and no liquid residue is generated in the valve portion in the middle of the flow path. Contamination can be prevented, specific components such as nucleic acids in a liquid sample can be extracted with high purity, and analysis can be performed with high accuracy.
[0087]
Alternatively, according to the present invention, the nucleic acid of the pathogen and the nucleic acid in leukocytes can be extracted and analyzed from the same whole blood sample, so that the presence or absence of infection by the pathogen can be confirmed, and at the same time, administration of the drug from the patient's genome information The effect can be predicted and the optimal drug can be selected. In particular, since a very small amount of whole blood that is surplus at the time of pathogen nucleic acid extraction is used for predicting the drug administration effect, the burden on the patient at the time of blood collection is small.
[0088]
Thereby, gene diagnosis can be performed with high accuracy and high efficiency.
[Brief description of the drawings]
FIG. 1 is an overall configuration diagram of a gene analyzer according to the present invention.
FIG. 2 is a block diagram of an analysis disk according to the present invention.
FIG. 3 is a configuration diagram of a flow path section according to the present invention.
FIG. 4 is an explanatory diagram showing a procedure of an analysis operation according to the present invention.
FIG. 5 is an explanatory diagram showing each operation of analysis according to the present invention and the correspondence between each operation and each drawing.
FIG. 6 is an operation explanatory diagram of the flow path section during the serum separation operation according to the present invention.
FIG. 7 is an operation explanatory diagram of the flow path section during the serum separation operation according to the present invention.
FIG. 8 is an operation explanatory diagram of the flow path section during the serum separation operation according to the present invention.
FIG. 9 is an operation explanatory diagram of the flow path section during the mixing operation of serum and lysate according to the present invention.
FIG. 10 is an operation explanatory diagram of the flow path part during the mixing and reaction operation of serum and lysate according to the present invention.
FIG. 11 is an operation explanatory diagram of the flow path section during the additional liquid combining operation according to the present invention.
FIG. 12 is an explanatory diagram of the operation of the flow path section during a cleaning operation according to the present invention.
FIG. 13 is an explanatory diagram of the operation of the flow path section during a cleaning operation according to the present invention.
FIG. 14 is an operation explanatory view of the flow path section during the cleaning operation according to the present invention.
FIG. 15 is an explanatory diagram of the operation of the flow path during the eluent flow operation according to the present invention.
FIG. 16 is an explanatory diagram of the operation of the flow path during the elution and eluent quantitative holding operation according to the present invention.
FIG. 17 is an explanatory diagram of the operation of the flow path during the amplification operation according to the present invention.
FIG. 18 (a) is a cross-sectional view of the reagent inlet and vent of each reagent container according to the present invention. (B) It is sectional drawing of the reagent inlet and the vent of each reagent container by this invention.
FIG. 19 is a circuit diagram of a positioning mechanism according to the present invention.
FIG. 20 is a timing chart of the positioning operation according to the present invention.
FIG. 21 is a block diagram of an analysis disk according to the present invention.
FIG. 22 is a configuration diagram of a flow path section according to the present invention.
FIG. 23 is an explanatory diagram showing a procedure of an analysis operation according to the present invention.
FIG. 24 is an explanatory diagram showing each operation of analysis according to the present invention and the correspondence between each operation and each drawing.
FIG. 25 is an explanatory view of the operation of the flow channel during the leukocyte lysis operation according to the present invention.
FIG. 26 is an explanatory view of the operation of the flow channel during the leukocyte lysis operation according to the present invention.
FIG. 27 is an explanatory diagram of the operation of the flow path portion during the mixing operation of the dissolved mixed solution and the binding solution according to the present invention.
FIG. 28 is an explanatory diagram of the operation of the flow path portion during the mixing operation of the dissolved mixed solution and the binding solution according to the present invention.
FIG. 29 is an explanatory diagram showing the procedure of an analysis operation according to the present invention.
FIG. 30 is an explanatory diagram of the operation of the flow path during serum separation and leukocyte lysis operation according to the present invention.
FIG. 31 is an explanatory diagram of the operation of the flow channel during serum separation and leukocyte lysis operation according to the present invention.
FIG. 32 is an overall configuration diagram of another gene analyzer according to the present invention.
FIG. 33 is an explanatory diagram showing another procedure of the analysis operation according to the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Gene analyzer, 2 ... Analysis disk, 11 ... Motor, 12 ... Holding disk, 13 ... Perforation machine, 14 ... Upper optical device, 15 ... Lower optical device , 16 ... Position detector, 20 ... Upper cover, 30 ... Flow path part, 121 ... Projection, 122 ... Holding disk optical window, 210 ... Sample injection port, 220 ... Reagent inlet, 221 ... vent, 301 ... nucleic acid binding member, 310 ... sample container, 316 ... serum capillary, 410 ... mixing part, 420 ... reaction vessel, 450 ..Detection container, 710 ... positioning hole

Claims (5)

A chemical analysis including a structure that is rotatably supported, and a structure that includes a capture unit that captures a specific chemical substance in a sample, and a plurality of reagent containers that hold liquid that flows through the capture unit. In the device
An eluent holding part for holding the eluent after elution of the specific chemical substance from the trapping part, a waste liquid discarding channel for discarding liquid other than the eluent from the eluent holding part, and an eluent from the eluent holding part An eluent disposal channel for discarding a part of the eluent disposal channel, and a connecting portion between the eluent holding unit and the eluent disposal channel is provided on the outer peripheral side of the innermost circumferential portion of the eluent disposal channel. Characteristic chemical analyzer. A chemical analysis including a structure that is rotatably supported, and a structure that includes a capture unit that captures a specific chemical substance in a sample, and a plurality of reagent containers that hold liquid that flows through the capture unit. In the device
An eluent holder that holds the eluent after elution of the specific chemical substance from the capture unit and a detection reagent container that supplies a detection reagent to the eluent holder are provided to control the flow of the detection reagent A detection reagent control unit is provided upstream of the detection reagent outlet for supplying the detection reagent to the eluent holding unit, and a part of the eluent is discarded from the eluent holding unit. A chemical analyzer characterized in that a flow path is provided and a detection reagent is caused to flow into an eluent holding part after a part of the eluent is discarded from the eluent disposal flow path. The chemical analysis apparatus according to claim 2, wherein the detection reagent control unit is an openable vent and an opening mechanism. The chemical analysis apparatus according to claim 2, wherein the detection reagent control unit is a reagent dispenser. A rotating structure that is rotatably supported, and a chemical structure that includes a capturing unit that captures a specific chemical substance in a sample, and a plurality of reagent containers that hold liquid that flows through the capturing unit. In the analyzer
An eluent holding part for holding the eluent after passing through the capturing part and an outflow channel for flowing out the liquid in the eluent holding part;
An outflow passage inlet that is a connection portion between the eluent holding portion and the outflow passage is provided on an inner peripheral side from an outflow passage outlet that is the other end of the outflow passage, and the trapping is performed during the rotation of the rotating structure. After the reagent flowing through the part flows through the outflow channel, the rotating structure stops once and then rotates again, and after stopping again, the eluent flows through the capturing part. .

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