JP2005180983A - Chemical analyzer and structure for chemical analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple chemical analyzer capable of grasping soundness on each treating process of a reagent or a sample in a cartridge, and extracting a specific chemical substance in a liquid sample with high accuracy. <P>SOLUTION: This chemical analyzer is equipped with a storage part for storing this structure, into which the sample is injected, storing the reagent to be reacted with the sample, and having a reaction region where the sample and the reagent are made to react, and a detection mechanism of the sample after reaction. The structure is equipped with a reagent storage part for storing the reagent, a reagent passage where the reagent flows, a sample passage where the sample flows, the reaction region connected to the reagent passage and the sample passage, and a chemical substance holding container, for holding the chemical substance to be mixed with the reagent, together with the sample in the reaction region. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a chemical analyzer and a chemical analysis structure.

  As a method for extracting DNA from a liquid sample containing DNA, Japanese Patent Publication No. 2003-502656 discloses a microstructure and a method for examination using amplification. In this apparatus, the DNA mixed solution is passed through a glass filter as an inorganic substrate, and then the washing solution and the eluent are passed through to collect only the DNA. 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. By opening a valve provided in a fine flow path connecting each reagent reservoir and the glass filter, each reagent flows under the action of centrifugal force and passes through the glass filter.

  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. In the integrated fluid operation cartridge, when each reagent is fed by a pump, the reagent passes through the capture component by opening a valve or the like provided in a fine channel connecting each reagent chamber and 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.

  As a method for quantifying DNA from a sample using an internal control, US Pat. No. 6,0015,722 discloses an aloe identification method using PCR. In this method, aloe DNA and internal control DNA are simultaneously extracted from a sample containing DNA as an internal control and amplified by PCR, whereby the aloe DNA is quantified by comparison with a detection signal from the internal control DNA.

Special table 2003-502656 gazette

Special table 2001-527220 gazette US 6001572

  However, in the structure described in JP-T-2003-502656 and the form described in JP-T-2001-527220, in each of the plurality of steps in which the sample reacts with the reagent and flows down, whether each process has been carried out easily or appropriately. It is difficult to know whether. For example, when analyzing samples processed using these devices, it is easy to determine whether the analysis result DATA is due to defects in the processing process, based on the characteristics of the sample itself, or due to other environmental factors. It is difficult to grasp.

  In the Aloe identification method using PCR described in US Pat. No. 6,0015,722, which is the third prior art, the DNA as the internal control is directly mixed into the sample, so that the internal control DNA is easily decomposed, particularly at a low concentration detection. In this case, the amount of internal control is unstable and the detection accuracy may be reduced. Furthermore, when detecting an RNA virus such as hepatitis C, it is desirable to use RNA as an internal control. However, RNA is more easily degraded than DNA, and the detection accuracy may further decrease.

  Accordingly, an object of the present invention is to provide a simple chemical analyzer that can solve at least one of the above-described problems and that extracts a specific chemical substance in a liquid sample with high accuracy. Another object is to provide a structure for chemical analysis.

  The present invention for solving the above-described problems has, for example, the following forms.

In this configuration, the reagent is stored and a cartridge in which a flow path for supplying the reagent to the supplied sample is formed is accommodated. For example, it is preferable to store one batch of a plurality of reagents. These cartridges may include a plurality of cartridges such as a reagent cartridge and a test cartridge. For example, the analyzer includes a storage unit for a reagent cartridge including a reagent storage unit, a connection part of the reagent cartridge, and a test cartridge (preferably valveless) in which a flow path through which the plurality of reagents flow down is formed.
Specifically, it has the following forms.
(1) A sample is injected, a reagent to be reacted with the sample is stored, a housing unit that houses a structure having a reaction region in which the sample and the reagent react, and a detection mechanism for the sample after the reaction; The structure includes a reagent storage unit that stores a reagent, a reagent channel through which the reagent flows, a sample channel through which the sample flows, the reagent channel, and the sample flow A chemical analyzer comprising: the reaction region communicating with a path; and a chemical substance holding container for holding a chemical substance mixed with the reagent in the reaction region together with the sample.

By flowing together with the sample and detecting the chemical substance component in the sample component detection unit, it is possible to effectively grasp whether the processing step such as reaction in the structure has been carried out effectively. it can.
(2) Or, in (1), the structure is rotatably installed in the container, the sample is a substance containing a first nucleic acid, and the chemical substance is a second nucleic acid different from the sample. And a capture unit that captures the first nucleic acid of the sample and the second nucleic acid of the chemical substance, and the detection mechanism is disposed outside the structure. It is a chemical analyzer.

  Thus, the chemical substance can be an internal control reagent. Here, the internal control reagent is such that it is mixed with the sample, mixed with the reagent together with the sample, and the chemical substance is captured together with the nucleic acid in the sample by the capturing unit. For example, as described above, it may be a substance containing a nucleic acid different from the nucleic acid to be examined. By inspecting the components (such as nucleic acids) in the captured chemical substance, it is possible to determine whether each step in the structure has been properly executed, and to improve the reliability of the inspection results. it can.

Furthermore, by amplifying and detecting the nucleic acid of the internal control reagent together with the nucleic acid of the captured sample, for example, if the detection result of the sample is lower than expected with respect to the detection result of the internal control reagent which is already a known component It is possible to examine whether or not there is a defect in the process or the sample. On the other hand, when both detection results are higher or lower than expected, the influence on the process and the environment can be examined.
(3) Specifically, in the above (1) or (2), the chemical substance holding container is formed so as to communicate with the sample channel on the upstream side of the reaction region.

  The sample flows along with the sample and is introduced into the reaction region. By flowing down with the sample, the state of the sample can be grasped effectively. After that, the chemical component to be captured in the sample and the specific component in the chemical substance are both captured by the capturing unit.

  Alternatively, the chemical substance holding container is formed so as to communicate with the reagent flow channel upstream of the reaction region. It flows down the flow path together with the reagent and is introduced into the reaction region. By flowing down with the reagent, the state of the reagent can be effectively grasped.

Alternatively, a lysis reagent that dissolves cell walls of cells contained in the sample is stored in the reagent storage unit, and the chemical substance holding container communicates with the upstream side of the reagent flow path that the reagent storage unit communicates with. Features.
(4) In any one of the above-described embodiments, the moisture absorbent holding unit that holds the moisture absorbent is in communication with the flow path between the chemical substance holding container and the reagent storage unit. Thereby, the hygroscopic change of the internal control reagent can be suppressed to form a highly storable structure, and an analyzer having excellent handling properties can be formed.
(5) In any one of the above-described embodiments, the structure includes a reagent cartridge including a reagent storage unit that stores a reagent, a connection unit to which the reagent cartridge is connected, a reagent channel through which the reagent flows, A test cartridge having a sample channel through which a sample flows, the reagent channel, and the reaction region communicating with the sample channel can be provided.

  According to the present invention, it is possible to grasp the soundness of each processing step of a reagent or a sample in a cartridge, and to provide a simple chemical analyzer that extracts a specific chemical substance in a liquid sample with high accuracy. be able to.

Embodiments of the present invention will be described below. In addition, this invention is not limited to the form as described below, and does not inhibit the change based on a well-known technique etc.

  With reference to FIGS. 1-17, the example of a genetic test apparatus is demonstrated as an analyzer of one Example by this invention.

  FIG. 1 is an overall configuration diagram of a genetic test apparatus according to the present invention. The gene analyzer 1 includes a holding disk 12 rotatably supported by a motor 11, a plurality of test modules 2 arranged on the holding disk 12, a punch 13 for controlling the flow of liquid, and heating. A device 14 and a detection device 15 are provided. The operator prepares the test module 2 for each test item, attaches it to the holding disk 12, and activates the genetic test apparatus 1.

  FIG. 2 is a configuration diagram of the inspection module 2. The inspection module 2 is configured by mounting a reagent cartridge 20 in which a transparent reagent cartridge cover 22 is joined to a reagent cartridge body 21 on an inspection cartridge 30 in which a transparent inspection cartridge cover 32 is joined to an inspection cartridge body 31.

  Each reagent is dispensed in a predetermined amount in each reagent container 220, 230, 240, 250, 260, 270, 280, 290 in advance. Reagent outlets 221, 231, 241, 251, 261, and 271 communicated with the respective reagent containers are provided on the back surface (FIG. 3) of the reagent cartridge 20. The reagent cartridges 321, 331, 341, 351, 361, and 371 are connected to the reagent inlets of the test cartridge 30. When the reagent cartridge 20 is mounted on the test cartridge 30, each reagent container communicates within the test cartridge through each reagent outlet and each corresponding reagent inlet.

  As an example of the present embodiment, specifically, for example, a structure in which a sample is introduced, holds a reagent that reacts with the sample, and reacts with the sample is stored in the first reagent storage that stores the first reagent. A reagent cartridge 20 having a section, a connecting portion to which the reagent cartridge 20 is connected, a first reagent channel through which the first reagent flows, a sample channel through which the sample flows, the reagent channel, and the A test cartridge having the reaction region in communication with the sample channel, a second reagent storage unit for storing a second reagent, and a second reagent supplied to the sample after reaction in the reaction region. The chemical substance holding container is held by the test cartridge 30.

  The reagent cartridge may be stored in the storage unit after being held at a temperature different from that of the test cartridge. As a specific example, the test cartridge is stored in the storage unit after being held at a temperature lower than that of the reagent cartridge.

  The inspection module is rotatably arranged, the pre-inspection object includes a nucleic acid, and the flow path cartridge holds a capturing unit that captures the nucleic acid of the inspection object and the captured nucleic acid. Holding part.

  Thereby, in particular, by using a mechanism for mounting the reagent cartridge 20 and the common flow path inspection cartridge 30 corresponding to the target inspection according to the inspection item, the reagent needs to be handled with care (temperature management etc.). Even if a defect occurs in the reagent, it is sufficient to use a reagent cartridge that does not have a defect only in the reagent cartridge 20, and the inspection cartridge 30 that is a flow path cartridge need not be changed, and an efficient inspection can be performed. In that case, the internal control can be effectively used, so that the reliability of the inspection can be improved.

  Further, the test cartridge 30 can be used as it is according to the object, and only the corresponding reagent cartridge 20 can be replaced, and an analyzer that can reduce stock space and cost can be configured.

  In addition, it is preferable to configure a valveless structure in which the inside of the reagent cartridge 20 communicates with the inside of the inspection cartridge when performing analysis. Specifically, in a state where the reagent cartridge and the inspection cartridge are mounted, a reagent container having the reagent cartridge 20 and another reagent container of the reagent cartridge are formed in the inspection cartridge 30. It arrange | positions so that the space connected via may be formed.

The cartridge also has its own characteristics.
The chemical analysis structure cartridge includes a reagent cartridge 30 including a reagent storage unit that stores a plurality of reagents, a connection unit to which the reagent cartridge 30 is connected, a reagent flow path through which the plurality of reagents flow down, It is characterized by comprising a test cartridge 20 having a sample channel through which a test object flows, and a reaction region where the reagent and the test object react.

  A sample introduction part containing a specific chemical substance, a reagent storage part for storing a reagent to be reacted with the sample, a reagent flow path through which the reagent flows, a sample flow path through which the sample flows, the reagent flow path, and the The reaction region that communicates with the sample flow path, and a capture unit that captures the chemical substance in the sample reacted in the reaction region, and includes an internal control reagent container containing a chemical substance different from the sample, The internal control reagent is a chemical analysis structure characterized in that the internal control reagent is mixed with the reagent together with the sample in the reaction region, and is captured together with the sample at a capture unit.

  This individual cartridge also has a feature.

  For example, an internal control can be provided on the test cartridge (FIG. 19).

  In this case, the test cartridge is configured to be connectable to a reagent structure having a reagent storage unit for storing a reagent, and includes a connection unit to which the reagent of the reagent storage unit is supplied and a sample containing a specific chemical substance. An injection port for injection, a separation unit for separating a separated sample containing a gene to be examined from a sample injected from the injection port, a first reagent supplied from the first connection unit, and the separated sample. A first reaction part that is introduced and reacted, a gene capture part that captures the gene from the reacted separated sample, and a cleaning reagent that leads the cleaning reagent supplied from the second connection part to the gene capture part A reagent flow path, an elution reagent flow path for guiding the elution reagent supplied from the third connection section to the gene capture section, and a holding section for holding the eluted gene, The first reaction part An internal control reagent container that is introduced together with the separated sample and the first reagent, is captured in the gene capturing unit together with the gene, and holds a chemical substance that is co-held with the gene in the holding unit. This is a characteristic inspection cartridge (FIG. 19).

  Note that detection may be performed by a detection mechanism in the holding unit.

  Further, the internal control reagent can be held in the reagent cartridge.

  In that case, a reagent cartridge capable of constituting a chemical analysis cartridge that holds an introduction part of a sample containing a specific chemical substance and a reagent that captures the specific chemical substance in the introduced sample and reacts with the sample. Can be connected to a test cartridge 20 having a reagent flow channel through which a reagent flows down, a sample flow channel through which a sample containing a specific chemical substance flows down, and the reaction region where the reagent flow channel and the sample flow channel communicate with each other A connection part formed in the container, a reagent storage part that stores the reagent, and a flow path that guides the stored reagent to the test cartridge 20, and is introduced into the reaction region through the connection part together with the reagent. It is a reagent cartridge characterized by having an internal control reagent container containing a chemical substance (FIGS. 10-17).

  The reagent cartridge and the test cartridge described above may be used as a pair with a cartridge having an internal control reagent container on one side and the other having no internal control reagent container.

  Further, the reagent cartridge may be formed on the inspection cartridge.

  Specifically, for example, an injection port for injecting a sample into the inspection cartridge is provided, and the injection port is arranged to be covered with the reagent cartridge when viewed from the stacking direction when the reagent cartridge is mounted. Thereby, it is possible to prevent the sample from diffusing and flowing out from the inlet when the cartridge rotates.

  Further, specifically, a connection part to which the reagent cartridge 20 can be connected, a reagent channel through which the reagent flows, a sample channel through which the sample flows, and the reaction in which the reagent channel and the sample channel communicate with each other. And an internal control reagent container in the reagent flow path formed in the reagent cartridge located upstream of the reaction region.

  Another form is a structure for chemical analysis that holds an introduction part of a sample containing a specific chemical substance and a reagent that captures the specific chemical substance in the introduced sample and reacts with the sample. The structure includes a dissolution reagent holding container that holds a dissolution reagent and a dissolution reagent holding container that holds a dissolution reagent, and the dissolution reagent holding container can be provided downstream of the dissolution reagent holding container.

  Alternatively, it may be a chemical analysis structure in which a moisture absorbing member is provided between the dissolved reagent holding container and the dissolved reagent holding container.

  FIG. 5 shows the main parts of the longitudinal sectional views of the reagent cartridge 20 and the test cartridge 30 in the AA section shown in FIGS. 2 and 4. FIG. 6 shows the main part of the test cartridge 30 mounted on the test cartridge 30. The longitudinal cross-sectional view corresponding to AA part is shown. A reagent cartridge protective sheet 23 is adhered to the lower surface of the reagent cartridge 20 in order to prevent leakage and evaporation of the reagent stored in advance in the reagent cartridge 20, and the inner surface of the inspection cartridge 30 is attached to the upper surface of the inspection cartridge 30. In order to prevent contamination, the inspection cartridge protection sheet 33 is adhered.

  The operator peels off the reagent cartridge protection sheet 23 and the inspection cartridge protection sheet 33 and attaches the reagent cartridge 20 to the inspection cartridge 30. The protrusions (for example, 269 in FIG. 5) constituting the reagent outlets are fitted with the reagent inlets to position both cartridges, and the reagent does not leak out of the test cartridge. Alternatively, an adhesive may be attached to the inspection cartridge cover 32 to which the inspection cartridge protective sheet is adhered, thereby adhering the joint surface of the reagent cartridge to prevent the reagent from leaking.

  Note that the protrusion provided on the reagent cartridge (for example, 269 in FIG. 5) may be provided on the inspection cartridge side.

Thus, it is characterized by having an uneven structure.
The reagent cartridge 30 includes a protrusion having a flow path communicating with the reagent container, and the test cartridge accommodates the protrusion and includes a concave inlet portion having a flow path corresponding to the flow path. . In addition, the combination formed in the cartridge on the opposite side to the said protrusion part and an inlet_port | entrance part may be sufficient.

The viral nucleic acid extraction and analysis operations when whole blood is used as a sample will be described below. FIG. 7 shows an operation procedure of the genetic test apparatus of the present invention by an operator, and FIGS. 8 and 9 show an operation procedure in the genetic test apparatus.
The operator injects whole blood collected by a vacuum blood collection tube or the like into the sample container 310 from the sample injection port 301 of the inspection cartridge 30 (FIG. 10), and the reagent cartridge protection sheet 23 and the inspection cartridge protection sheet shown in FIG. After peeling 33, the reagent cartridge 20 is mounted on the inspection cartridge 30 (FIG. 2). At this time, the sample inlet 301 of the test cartridge 30 is blocked by the reagent cartridge 20, so that the sample will not leak from the test cartridge 30 thereafter. Alternatively, a sample vent hole 313 is passed through the reagent cartridge 20 (FIGS. 2 and 3), and a filter that allows air to pass but does not allow the sample and its mist to pass is attached so that the sample can be vented when it flows. May be.

  When the necessary number of analysis modules 2 assembled in this manner are mounted on the holding disk 12 in FIG. 1 and the genetic test apparatus 1 is operated, viral nucleic acid is extracted from the whole blood, and finally the nucleic acid is detected. Is done.

Below, the flow state of the liquid in each operation | movement in the genetic test | inspection apparatus 1 is shown in FIGS.
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. 11). The whole blood disposal container 315 is provided with a whole blood disposal vent channel 318, and air can enter and exit from the reagent cartridge vent 202 through the test cartridge vent 302. 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.

  When the rotation is further continued, the whole blood 501 is separated into blood cells and serum or plasma (hereinafter referred to as “serum”) (centrifugation), the blood cells 502 move to the blood cell storage container 311 on the outer peripheral side, and the serum quantification container 312 has the serum 503. (FIG. 12).

  During the series of serum separation operations, the air holes 222, 232, 242, 252, 262, and 272 of the reagent containers in the reagent cartridge 20 are covered with the reagent cartridge cover 22 (FIG. 5) so that air does not enter. It has become. 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. 12, a flow path structure (return flow paths 223, 233, 243, 253, 263, 273) 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.

  When the serum separation operation is completed by rotating for a predetermined time, the analysis module 2 stops, and a part of the serum 503 in the serum quantitative container 312 flows into the capillary 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.

  Thereafter, the perforator 13 opens holes one by one in the upstream of each reagent container, rotates the motor 11, and causes each reagent to flow with centrifugal force.

  The operation after serum separation is shown below.

  In the lysing solution container 220, 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 solution vent hole 222, the solution 521 passes from the solution container 220 through the solution return channel 223 by the action of centrifugal force, and the hygroscopic material 291 is removed. Then, it flows into the internal control container 290, and the solution flows into the mixing unit 410 while mixing with the internal control 590. The internal control is a nucleic acid or a synthetic product containing nucleic acid, and is preferably lyophilized so that it can be stored for a long period of time. Alternatively, it may be in a frozen state. In some cases, a dry product is used. Therefore, when the solution flows into the internal control container 290, it mixes and flows out while dissolving the internal control 590.

  The hygroscopic material 291 is provided between the solution container 220 and the internal control container 290 so that the internal control 590 does not absorb the moisture of the solution 521. The internal control container 290 may be configured to communicate with a flow path through which the solution flows through a communication path. Hygroscopic materials include silica gel structures, porous structures using other materials or structures that make up fine flow paths such as fiber filters, and protrusions made of silicon or metal made by etching or machining. Use it.

  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 and is mixed in the mixing unit 410 with the dissolved solution in which the internal control flowing in simultaneously is dissolved (FIG. 13). The mixing unit 410 has 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.

  Serum and lysate are mixed in the mixing section 410 and flow into the reaction container 420 (FIG. 14). The reaction container 420 is provided with a reaction container ventilation channel 423, and air can enter and exit from the reagent cartridge ventilation hole 202 through the inspection cartridge ventilation hole 302. 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.

  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.

  In the reaction container 420, the mixed serum and the lysate react. Since the liquid level in the reaction vessel 420 after the mixed solution of serum and lysate flows into the reaction vessel 420 is on the outer peripheral side of the innermost peripheral portion (radius position 604) of the reaction solution flow path 421, the reaction solution The innermost peripheral portion of the flow path 421 cannot be exceeded, and the mixed liquid is held in the reaction vessel 420 during rotation.

The lysing solution functions to dissolve the membrane from viruses, bacteria, and the like in the serum to elute the nucleic acid, but it is preferable to further promote the adsorption of the nucleic acid to the nucleic acid binding member 301. Similarly, adsorption of the dissolved internal control 590 to the nucleic acid binding member 301 is promoted. As such a reagent, for example, guanidine hydrochloride is used for elution and adsorption of DNA, guanidine thiocyanate is used for RNA, and a porous material such as quartz or glass or a fiber filter is used as a nucleic acid binding member. After the lysate is held in the reaction vessel 420, the motor 11 is stopped, the perforator 13 opens a hole in the additional liquid vent 232 for supplying air to the additional liquid container 230, and the motor 11 is rotated again. The additional liquid 531 flows from the additional liquid container 230 through the additional liquid return flow path 233 into 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. 15). . When the liquid level reaches the innermost peripheral part (radius position 604) of the reaction liquid flow path 421, the mixed liquid flows out beyond the innermost peripheral part of the reaction liquid flow path, and passes through the converging flow path 701 to the nucleic acid binding member 301. Flows in. As the additional liquid, for example, the above-described dissolving liquid may be used.

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.
When the mixed solution of lysate and serum passes through the nucleic acid binding member 301 in this way, the target nucleic acid in the serum and the nucleic acid as the internal control are adsorbed to the nucleic acid binding member 301, and the liquid flows into the eluent recovery container 390 .

  The eluent recovery container 390 is provided with an eluent recovery container vent flow path 394 through which air can enter and exit from the reagent cartridge vent hole 202 through the test cartridge vent hole 302. The waste liquid 391 after passing through the nucleic acid binding member 301 is once held in the eluent recovery container 390 for the waste liquid return channel 393 as in the case of the mixing container 420, but the eluent recovery container 390 is compared with the amount of waste liquid. Therefore, the waste liquid flows over the innermost peripheral side of the waste liquid return flow path 393 and flows out to the waste liquid storage container 402 through the waste liquid outflow path 399 (FIG. 16).

  Next, when the motor 11 is stopped and the perforator 13 opens a hole in the first cleaning liquid vent 242 for supplying air to the first cleaning liquid container 240, and then the motor 11 is rotated again, the centrifugal force acts. The first cleaning liquid flows from the first cleaning liquid container 240 into the nucleic acid binding member 301 via the first cleaning liquid return flow path 243 and the merge flow path 701 to wash unnecessary components such as proteins adhering to the nucleic acid binding member 301. As the first cleaning liquid, for example, the above-described dissolution liquid or a liquid with a reduced salt concentration may be used.

  The waste liquid after washing flows out to the waste liquid storage container 402 through the eluent recovery container 390, like the above-described mixed liquid.

The same cleaning operation is repeated several times. For example, following the first cleaning liquid, with the motor stopped, the perforator 13 opens a hole in the second cleaning liquid vent hole 252 for supplying air to the second cleaning liquid container 250, and rotates the motor 11 again, so that the nucleic acid binding member Wash away unnecessary components such as salt adhering to 301. As the second cleaning liquid, for example, ethanol or an aqueous ethanol solution may be used.
Similarly, a hole is made in the lid of the third cleaning liquid vent hole 262 for supplying air to the third cleaning liquid container 260. The third cleaning liquid flows directly into the eluent recovery container 390 and cleans components such as salt attached to the eluent recovery container 390. As the third cleaning liquid, for example, sterilized water or an aqueous solution whose pH is adjusted to 7 to 9 may be used.

  After washing the nucleic acid binding member 301 and the eluent recovery container 390 in this way, the process proceeds to the nucleic acid elution step.

  That is, with the motor stopped, a hole is made in the lid of the eluent vent hole 272 for supplying air to the eluent container 270 by the punching machine 13, the motor 11 is rotated again, and the eluent 571 flows through the nucleic acid binding member 301. (FIG. 17). The eluent is a liquid that elutes nucleic acid from the nucleic acid binding member 301, and water or an aqueous solution adjusted to pH 7 to 9 may be used. In particular, it is desirable to heat to 40 degrees or more in order to facilitate elution. For heating, the heating device 14 of FIG. 1 may be used, and light may be irradiated from above the eluent recovery container 390. The nucleic acid collected in this manner is taken out and injected into a detection device, and the nucleic acid is amplified and detected.

  Alternatively, amplification and detection in the eluent recovery container 390 can be performed by using an amplification liquid as the eluent 571 as described below. That is, in a state where the motor is stopped, a hole is formed in the amplification liquid vent hole 272 for supplying air to the amplification liquid storage container 270 by the perforator 13 and the motor 11 is rotated again to flow the amplification liquid 571 (FIG. 17). The amplification solution 571 passes through the enzyme reagent container 280 containing the enzyme reagent, enters the enzyme reagent mixing container 380 together with the enzyme reagent 580, and once the amplification reagent mixes with the enzyme reagent, passes through the nucleic acid binding member 301, Elute. The enzyme reagent 580 is preferably lyophilized so that it can be stored for a long time. That is, when the amplification solution 571 flows into the enzyme reagent container 280, the enzyme reagent 580 is dissolved and flows out, and flows into the enzyme reagent mixing container 380. It is desirable to provide a hygroscopic material 281 between the eluent container 270 and the enzyme reagent container 280 so that the enzyme reagent does not absorb the moisture of the amplification liquid 571 during storage. Hygroscopic materials include silica gel structures, porous structures using other materials or structures that make up fine flow paths such as fiber filters, and protrusions made of silicon or metal made by etching or machining. Use it.

  The amount of the liquid from which the nucleic acid has been eluted is smaller than the volume of the eluent recovery container 390, cannot exceed the innermost peripheral side of the waste liquid return channel 393, and is retained in the eluent recovery container. The amplification solution is a reagent for amplifying and detecting nucleic acid, and includes deoxynucleoside triphosphate, a fluorescent reagent, and the like. Depending on the amplification method, the heating device 14 is used to irradiate light from above the eluent recovery container 390 for heating, and the nucleic acid is amplified in the eluent recovery container 390.

  Next, the detection device 15 is moved onto the eluent collection container 390 to detect, for example, the amount of fluorescence emitted from the target nucleic acid and the internal control nucleic acid.

  Internal controls are pre-quantified nucleic acids or synthetic products containing nucleic acids, and are extracted, amplified, and detected using exactly the same reagents, cartridges, and testing equipment as the extraction, amplification, and detection of target nucleic acids in serum. Therefore, if the extraction / amplification / detection steps are functioning normally, a signal such as predetermined fluorescence or absorbance can be detected from the internal control. On the other hand, if the signal intensity is low or not detected at all, it can be seen that there is an abnormality in any of the extraction, amplification, and detection processes due to a defect in the reagent, cartridge, inspection device, or the like. Alternatively, the concentration of the target nucleic acid can be quantitatively evaluated by comparing the detection signal of the target nucleic acid with the detection signal of the internal control that has been quantified in advance.

  According to the present embodiment, a reagent that cannot be stored in a liquid form for a long period of time can be stored in a reagent cartridge in a lyophilized state, so that it can be stored for a long period of time, and a test can be easily performed without adjusting the reagent.

  Alternatively, according to the present invention, the internal control nucleic acid is stored in the reagent cartridge in advance, and the nucleic acid extraction operation or the amplification / detection operation is performed simultaneously with the target nucleic acid in the serum. The target nucleic acid concentration of the sample can be determined by guaranteeing the sexiness or comparing with the internal control.

  This eliminates the need for reagent dispensing and eliminates the risk of reagent contamination due to inadequate work. In addition, there is no need to provide a valve for controlling the flow of each reagent in the middle of the flow path, no liquid residue is generated in the valve section in the middle of the flow path, and contamination by the reagent in the previous process can be prevented. Specific components such as nucleic acids therein can be extracted with high purity and analyzed with high accuracy.

  In addition, according to the present invention, since the reagent cartridge is supplied separately from the inspection cartridge, only the reagent cartridge is provided for each inspection item in accordance with the inspection item. Since the inspection cartridge can be used in common, the production cost can be reduced.

  Alternatively, only the reagent cartridge must be stored for each inspection item, and the number of inspection cartridges can be reduced, and the storage space can be reduced. In particular, when it is necessary to manage the temperature of the reagent, only the temperature of the reagent cartridge needs to be controlled, and the space required for temperature management can be reduced.

  Alternatively, even if a reagent failure occurs due to a power failure or the like, the test cartridge can be used independently, so it is economical to purchase only the reagent cartridge.

In particular, by providing a hygroscopic material between the liquid reagent container and the lyophilized reagent container, the lyophilized reagent is prevented from absorbing the moisture of the liquid reagent, and can be stored for a longer period of time.
In the embodiment shown in FIG. 2, an internal control container 290 is provided on the downstream side of the dissolution liquid container 220, and the dissolution liquid 521 flows into the internal control container 290 to dissolve the freeze-dried internal control 590. Although mixed with serum, the internal control may be held in a sample container 310 to which a sample such as whole blood is supplied or a blood cell storage container 311 from which a component of the sample such as whole blood is separated. At this time, as an internal control, a synthetic product containing a nucleic acid such as an artificial virus that is not degraded by a nucleolytic enzyme in blood is desirable.

  In the embodiment shown in FIG. 2, the hygroscopic material 291 is provided so that the internal control 590 does not absorb the moisture of the solution 521, but a valve may be provided instead of the hygroscopic material. This valve is for storing the cartridge for a long period of time, and preferably has a structure that is opened by pressurization or heating immediately before inspection. Alternatively, the strength may be weak enough to be released during the first centrifugal operation. Moreover, the container which accommodates a hygroscopic material may be arrange | positioned via the connection path with respect to the flow path through which a solution flows.

In the embodiment shown in FIG. 2, the hygroscopic material 291 is provided at one place, but a plurality of places may be provided between the solution container 220 and the internal control container 290 in order to improve the hygroscopic effect.
In the embodiment shown in FIG. 2, the internal control 590 is dissolved by flowing the dissolving solution 521 only once. However, the dissolving solution 521 is divided into a plurality of times and flows from the dissolving solution container 220, or a plurality of dissolving solution containers are provided. Dissolution may be performed reliably by providing a single solution and flowing a solution.
In the embodiment shown in FIG. 2, the internal control container 290 is provided on the downstream side of the dissolution liquid container 220. However, as in the other embodiment shown in FIG. 290, a hygroscopic material 291 provided therebetween, an internal control solution container 295 for holding the internal control solution 595 is provided further upstream of the internal control container 290, and an internal control solution hygroscopic material 296 is provided therebetween. ing.

  That is, the internal control 590 is once dissolved with the internal control solution 595 and then mixed with the solution 521.

  Since the solution 521 generally uses a chaotropic salt, the internal control 590 is difficult to dissolve. However, for example, sterilized water near pH 7 can be used as the internal control solution to facilitate dissolution.

  In these embodiments, the form in which the internal control container is formed inside the sample cartridge has been described. However, the internal control container may be formed so as to communicate with the sample flow path through which the sample is provided.

  In that case, it is preferably arranged so as to merge with the separated serum. Alternatively, it may be arranged so as to communicate with a region between a sample supply port such as whole blood and a sample component separation part.

  With such a configuration, it is possible to grasp the soundness of each processing step of the reagent or sample in the cartridge, and to provide a simple chemical analyzer that extracts a specific chemical substance in a liquid sample with high accuracy. be able to.

  Further, by adopting the form of the present embodiment in which the valve is not installed, the reagent dispensing operation becomes unnecessary, and there is no fear of contamination of the reagent due to inadequate work. In addition, there is no need to provide a valve for controlling the flow of each reagent in the middle of the flow path, no liquid residue is generated in the valve section in the middle of the flow path, and contamination by the reagent in the previous process can be prevented. Specific components such as nucleic acids therein can be extracted with high purity and analyzed with high accuracy.

  In the embodiment shown in FIG. 19, only the freeze-dried reagent is held on the test cartridge side. That is, the internal control container 473 is provided in a test cartridge located on the downstream side of the lysing solution container 220 provided in the reagent cartridge, and an enzyme reagent 472 that is a reagent used in the amplification process, particularly a reagent containing an enzyme, is provided in the reagent cartridge. 20 is provided in the inspection cartridge 30 located on the downstream side of the eluent container 471.

  In the embodiment shown in FIG. 10 and the like, an amplification reagent cartridge that needs to be stored at a low temperature is manufactured in the reagent cartridge 20, but as shown in FIG. 19, an enzyme reagent 472 that is a low-temperature reagent container is provided inside the test cartridge 30. The test cartridge 30 may be stored at a low temperature while being provided together with the control container 473 and preliminarily holding an amplification reagent such as an enzyme that needs to be stored at a low temperature. Only reagents that do not need to be stored at a low temperature are stored in the reagent cartridge 20, and amplification reagents other than the low-temperature reagents may be held in advance in the room temperature amplification reagent container. When a hole is made in a room temperature amplification reagent container vent for supplying air to the room temperature amplification reagent container with a perforator and the motor 11 is rotated, the room temperature amplification reagent flows out of the room temperature reagent container, and the cryogenic reagent in the cryogenic reagent container. And let it flow out.

  As described above, the test cartridge 30 in which the mixing channel is formed is provided with a reagent container for holding the reagent and is connected to the reagent cartridge 20 for storing the reagent. Thereby, the above-described effect can be obtained while suppressing an increase in the number of cartridges. For this reason, since it is a simple structure, it is easy to handle and a simple structure can be configured.

  For example, the test cartridge 30 includes a reagent storage unit that stores a reagent, and the test cartridge 30 is held at a temperature lower than that of the reagent cartridge 20 and then accommodated in the analyzer main body.

  For example, the test cartridge is refrigerated (about −20 ° C.), and an enzyme (used during amplification) is stored as a reagent. The reagent cartridge is held at a higher temperature, stored at room temperature (about 5-40 ° C.), and the extraction reagent, detection reagent: fluorescent dye, and the like are stored.

  According to the present invention, even if two types of reagents having different storage temperature conditions exist, it is not necessary to divide the reagent cartridge itself by simply manufacturing the reagent cartridge separately from the inspection cartridge, thereby reducing the manufacturing cost. Can be handled easily.

  According to the present invention, the dispensing operation of the reagent is unnecessary, and there is no concern about contamination of the reagent due to inadequate work. In addition, there is no need to provide a valve for controlling the flow of each reagent in the middle of the flow path, no liquid residue is generated in the valve section in the middle of the flow path, and contamination by the reagent in the previous process can be prevented. Specific components such as nucleic acids therein can be extracted with high purity and analyzed with high accuracy.

  Since the internal control container 473 is provided in the cartridge having such a configuration, it can greatly contribute to the implementation of a highly accurate inspection.

  Thus, these test cartridges 30 are stored in the analyzer after being held at a temperature lower than that of the reagent cartridge 20.

  For example, the reagent cartridge 20 has a smaller volume than the inspection cartridge 30. Further, the component, amount, or number of reagents stored in the test cartridge 30 can be smaller than the amount or number of reagents stored in the reagent cartridge 20.

  Further, as shown in the figure, the reagent cartridge 20 is characterized in a stacked arrangement. For example, the reagent cartridge 20 is installed facing the inspection cartridge 30.

  Specifically, a reagent cartridge having a higher holding temperature or room temperature storage is stacked on a refrigerated inspection cartridge.

  In addition, according to the present invention, the lyophilized reagent can be held independently of the liquid reagent, so that moisture absorption can be prevented.

In the embodiment shown in FIG. 20, the structure is such that all the reagents are held inside the test cartridge 3 without making the cartridge dedicated to the reagent independent. That is, in the test cartridge 3, the internal control container 290 that holds the internal control 590 is located downstream of the dissolution liquid container 220 that holds the dissolution liquid 521, and a hygroscopic material 291 is provided between the two containers. The moisture of the solution 521 is prevented from absorbing moisture.
Further, a hygroscopic material 292 may be provided on the downstream side of each reagent container to prevent mixing due to evaporation of the reagent between the reagents.

  According to the present invention, it is not necessary to manufacture the reagent cartridge and the inspection cartridge separately, the manufacturing cost can be reduced, and handling is simplified.

  In each of the above embodiments, the reagent is held in the cartridge. However, the reagent container may be dispensed using a pipetter or the like.

1 is an overall configuration diagram of a genetic test apparatus according to an embodiment of the present invention. It is a block diagram of the test | inspection module by one Example of this invention. It is a block diagram of the reagent cartridge by one Example of this invention. It is a block diagram of the test | inspection cartridge by one Example of this invention. It is sectional drawing of the reagent cartridge and test | inspection cartridge by one Example of this invention. It is sectional drawing of the test | inspection module by one Example of this invention. It is explanatory drawing which shows the operation procedure by one Example of this invention. It is explanatory drawing which shows the operation | movement procedure within the genetic diagnostic apparatus by one Example of this invention. It is explanatory drawing which shows the operation | movement procedure within the genetic diagnostic apparatus by one Example of this invention. It is operation | movement explanatory drawing of the reagent cartridge and analysis cartridge by one Example of this invention. It is operation | movement explanatory drawing of the reagent cartridge and analysis cartridge by one Example of this invention. It is operation | movement explanatory drawing of the reagent cartridge and analysis cartridge by one Example of this invention. It is operation | movement explanatory drawing of the reagent cartridge and analysis cartridge by one Example of this invention. It is operation | movement explanatory drawing of the reagent cartridge and analysis cartridge by one Example of this invention. It is operation | movement explanatory drawing of the reagent cartridge and analysis cartridge by one Example of this invention. It is operation | movement explanatory drawing of the reagent cartridge and analysis cartridge by one Example of this invention. It is operation | movement explanatory drawing of the reagent cartridge and analysis cartridge by one Example of this invention. It is a block diagram of the reagent cartridge by one Example of this invention. It is a block diagram of the reagent cartridge by one Example of this invention. It is a block diagram of the test | inspection cartridge by one Example of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Gene test | inspection apparatus, 2 ... Test | inspection module, 11 ... Motor, 12 ... Holding disk, 13 ... Punching machine, 14 ... Heating apparatus, 15 ... Detection apparatus, 20 ... Reagent cartridge, 30 ... Test cartridge.

Claims (12)

A sample is injected, a reagent to be reacted with the sample is stored, and a storage unit that stores a structure having a reaction region in which the sample and the reagent react with each other, and a detection mechanism for the sample after the reaction are provided. A chemical analyzer,
The structure includes a reagent storage unit that stores a reagent, a reagent channel through which the reagent flows, a sample channel through which the sample flows, the reaction channel that communicates with the reagent channel and the sample channel, A chemical substance holding container for holding a chemical substance to be mixed with the sample and the reagent in a reaction region;
The sample is a substance containing a first nucleic acid, the chemical substance is a substance containing a second nucleic acid different from the sample, and captures the first nucleic acid of the sample and the second nucleic acid of the chemical substance. A chemical analysis apparatus, wherein the detection mechanism is disposed outside the structure. The chemical analyzer according to claim 1, wherein the structure is rotatably installed in the housing portion. 2. The chemical analysis apparatus according to claim 1, wherein the chemical substance holding container is formed so as to communicate with the sample channel on the upstream side of the reaction region. 2. The chemical analyzer according to claim 1, wherein the chemical substance holding container is formed so as to communicate with the reagent flow channel upstream of the reaction region. 2. The chemical storage container according to claim 1, wherein a lysis reagent that dissolves a cell wall of a cell contained in the sample is stored in the reagent storage unit, and the chemical substance holding container communicates with an upstream side of the reagent flow path that the reagent storage unit communicates with. A chemical analysis device characterized by: 5. The chemical analysis apparatus according to claim 4, wherein a hygroscopic material holding unit for holding a hygroscopic material communicates with a flow path between the chemical substance holding container and the reagent storage unit. In claim 1,
The structure includes a reagent cartridge including a reagent storage unit for storing a reagent, a connection unit to which the reagent cartridge is connected, a reagent channel through which the reagent flows, a sample channel through which the sample flows, and the reagent flow And a test cartridge having the reaction region communicating with the sample channel, and the chemical substance holding container is held by the reagent cartridge. In claim 1,
The structure includes a reagent cartridge including a reagent storage unit for storing a reagent, a connection unit to which the reagent cartridge is connected, a reagent channel through which the reagent flows, a sample channel through which the sample flows, and the reagent flow A chemical analysis apparatus comprising: a test cartridge having a path and the reaction region communicating with the sample flow path, wherein the chemical substance holding container is formed in the test cartridge. A structure for chemical analysis,
A sample introduction part containing a specific chemical substance, a reagent storage part for storing a reagent to be reacted with the sample, a reagent flow path through which the reagent flows, a sample flow path through which the sample flows, the reagent flow path, and the A reaction region that communicates with the sample flow path, and a capture unit that captures the chemical substance in the sample reacted in the reaction region, and includes an internal control reagent container containing a chemical substance different from the sample, The structure for chemical analysis, wherein the internal control reagent is mixed with the sample and the reagent in the reaction region and captured together with the sample in the capture unit. A connection unit configured to be connectable to a reagent structure including a reagent storage unit for storing a reagent, and supplied with a reagent of the reagent storage unit;
An inlet for injecting a sample containing a specific chemical substance;
A separation unit for separating a separated sample containing a nucleic acid to be examined from a sample injected from the injection port;
A first reaction unit that introduces and reacts the first reagent supplied from the first connection unit and the separated sample;
A nucleic acid capture unit for capturing the nucleic acid from the reacted separated sample;
A cleaning reagent flow path for guiding the cleaning reagent supplied from the second connection section to the nucleic acid capturing section;
An elution reagent flow path for guiding the elution reagent supplied from the third connection section to the nucleic acid capture section;
A holding unit for holding the eluted nucleic acid,
An internal control reagent container that holds a chemical substance that is introduced into at least the first reaction unit together with the separated sample and the first reagent, captured in the nucleic acid capturing unit together with the nucleic acid, and retained in the retaining unit together with the nucleic acid. And an inspection structure characterized by comprising: Formed to be connectable to a test structure having a reagent flow path through which a reagent flows, a sample flow path through which a sample containing a specific chemical substance flows, and a reaction region where the reagent flow path and the sample flow path communicate with each other And a chemical reservoir introduced into the reaction region through the connecting portion together with the reagent. The connecting portion is configured to be connected to the test structure. A reagent structure comprising an internal control reagent container having a substance. A sample is injected, a reagent to be reacted with the sample is stored, and a storage unit that stores a structure having a reaction region in which the sample and the reagent react with each other, and a detection mechanism for the sample after the reaction are provided. A chemical analyzer,
The structure includes a reagent storage unit that stores a reagent, a reagent channel through which the reagent flows, a sample channel through which the sample flows, the reaction channel that communicates with the reagent channel and the sample channel, A chemical analysis apparatus comprising: a chemical substance holding container that holds a chemical substance mixed with the reagent arranged to communicate with the reagent flow path between a reaction region and the reagent storage unit.

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