Genetic testing device

Abstract

This invention provides a genetic testing device that can efficiently detect pre-disease states by examining the characteristics of specific regions of genes. [Solution] The gene testing device 1 comprises a sample container placement unit 2 for placing a sample container containing blood, a leukocyte separation unit 3 for recovering a first solution containing leukocytes from the blood using a leukocyte separation device 31, a concentration measurement unit 4 for optically measuring the concentration of leukocytes in the first solution, a reagent mixing unit 5 for mixing a cell membrane lysate with the first solution, adjusting the temperature, and then mixing in a probe reagent to prepare a second solution, a second solution processing unit 6 for reacting with the probe reagent to form a luminescent portion at a specific site of the gene present in the leukocytes in the second solution, an optical measurement unit 7 for chemiluminescence of the luminescent portion and detecting the amount of luminescence with a second photodetector, and a measurement processing unit 8 for calculating the characteristics of the specific site of the gene from the detection results of the concentration measurement unit 4 and the optical measurement unit 7.

Description

Technical Field

[0001] The present disclosure relates to a genetic testing device that optically examines specific sites of genes possessed by white blood cells contained in blood.

Background Art

[0002] Due to the increasing emphasis on health in recent years, it has been proposed that it is preferable to quickly detect not only the diagnosis of diseases but also the state called pre-disease. Pre-disease refers to a state that is approaching disease between health and disease. If it is found that a person is in a pre-disease state, for example, measures such as improving lifestyle habits can be taken. By doing so, the possibility of developing a disease can be reduced and a healthy state can be maintained.

[0003] Therefore, as a method for detecting the pre-disease state, it has been proposed and put into practical use to examine the characteristics of specific sites of genes. As a typical method of such examination, for example, the Hybridization Protection Assay (HPA) method is known.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] There is a need for a genetic testing device that can efficiently perform a method for detecting a pre-disease state by examining the characteristics of specific sites of genes, such as the HPA method.

Means for Solving the Problems

[0006] One embodiment of the gene testing apparatus of this disclosure is a gene testing apparatus for optically examining specific sites of genes in leukocytes contained in blood, comprising a sample container placement unit, a leukocyte separation unit, a concentration measurement unit, a reagent mixing unit, a second liquid processing unit, an optical measurement unit, and a measurement processing unit. The sample container placement unit is a part for placing a sample container containing blood obtained by blood collection as a sample. The leukocyte separation unit is a part for collecting blood from the sample container in the sample container placement unit, then separating leukocytes in the blood using a leukocyte separation device, and recovering a first liquid containing leukocytes as the main blood cells. The concentration measurement unit is a part for optically measuring the concentration of leukocytes in the first liquid, comprising a first light incidence means and a first light detection means. The reagent mixing unit is a part for preparing a second liquid by mixing a cell membrane lysate with the first liquid, adjusting the temperature, and then mixing in a probe reagent. The second liquid processing unit is the part that heats and cools the second liquid to react a specific site of the gene present in the leukocyte with the probe reagent, thereby forming a light-emitting part at the specific site of the gene present in the leukocyte in the second liquid. The optical measurement unit is the part that mixes hydrogen peroxide solution and an alkaline solution with the second liquid containing the gene on which the light-emitting part has been formed to cause the light-emitting part to chemiluminescent, and detects the amount of light emitted from the light-emitting part with a second photodetector. The measurement processing unit is the part that calculates the characteristics of the specific site of the gene present in the leukocyte from the detection results of the first detection unit and the detection results of the second detection unit. [Effects of the Invention]

[0007] According to the gene testing device described herein, which includes the above components and optically detects reactions based on the characteristics of specific regions of genes, it is possible to efficiently test the characteristics of specific regions of genes. It is possible. [Brief explanation of the drawing]

[0008] [Figure 1] This is a schematic block diagram showing the general configuration of an example of a gene testing device in this disclosure. [Figure 2] This is a schematic plan view showing an example of a leukocyte separation device. [Figure 3]This is a schematic plan view showing an example of the configuration of the flow channel and multiple holes in a leukocyte separation device. [Figure 4] This is a plan view showing region IV, enclosed by the dashed-dotted rectangle in Figure 3. [Figure 5] This is a simplified diagram showing an example of the general structure of a chromosome. [Modes for carrying out the invention]

[0009] The HPA method, a diagnostic test that can detect whether a person is in a pre-disease state, measures the degree of fatigue of telomeres, which are structures located at the ends of chromosomes. Figure 5 shows a simplified example of the schematic structure of a chromosome. Telomere 91 is a structure located at the end of deoxyribonucleic acid (DNA) on chromosome 90. Telomere 91 protects important genetic information within chromosome 90 and is a crucial structure that determines cellular aging, and is also thought to be related to age-related diseases. Measuring the fatigue level of telomere 91 is useful for detecting pre-disease states.

[0010] When measuring the fatigue level of telomere 91, the length of a region called the G-tail 92, located at the end of telomere 91, is measured. When the G-tail 92 shortens due to unhealthy lifestyle habits, the individual becomes more susceptible to disease. Conversely, improving lifestyle habits can lengthen the G-tail 92. Therefore, measuring telomere length or G-tail length, as characteristics of specific gene regions such as telomere 91 or the G-tail 92 of telomere 91, is useful as a genetic test.

[0011] The HPA method is known for measuring the length of the G-tail, specifically the length of the G-tail sequence. The HPA method involves hybridizing multiple labeled probes complementary to the telomere repeat sequences that make up the G-tail to the G-tail 92, causing the non-radioactive labeled substance bound to the labeled probes (hereinafter also simply called probes) to chemiluminescent, and measuring the length of the G-tail sequence using the amount of chemiluminescence as an indicator. Hybridization is the process by which nucleic acid molecules (DNA or ribonucleic acid (RNA)) form a complementary complex, and is also called molecular cross-pollination. Hybridization, which uses the complementarity of bases to create double-stranded molecules from nucleic acids of different origins, is called hybridization.

[0012] In the HPA method, oligomers labeled with a non-radioactive labeling substance are used as labeled probes, and the chemiluminescence from the non-radioactive labeling substance when this labeled probe hybridizes with the target of detection, such as DNA, is detected. In this process, to distinguish between the hybridized probe and the free probe that has not hybridized, the labeling substance of the free probe is selectively hydrolyzed to inactivate it. Therefore, with the HPA method, the target G-tail 92 can be detected in a short time without using complicated procedures, and the length of the G-tail sequence, i.e., the length of G-tail 92, can be measured using the amount of chemiluminescence of the labeling substance as an indicator.

[0013] In the gene testing device disclosed herein, when examining the characteristics of a specific region of a gene, the HPA method is used to detect the amount of chemiluminescence corresponding to the length of the G-tail, which is a characteristic of the specific region, and the characteristics of the specific region are examined.

[0014] The gene testing device disclosed herein will be described below with reference to the drawings. Parts with similar configurations and functions are denoted by the same reference numerals, and redundant explanations are omitted in the following description. The drawings are for illustrative purposes only.

[0015] In addition, the drawings include diagrams to which a right-handed XYZ coordinate system is appended for convenience. In the following description, the +Z direction is adopted as vertically upward (also simply referred to as upward). The vertically downward direction is also expressed as the -Z direction. The direction opposite to the X direction is also expressed as the -X direction. The direction opposite to the Y direction is also expressed as the -Y direction.

[0016] FIG. 1 is a block diagram schematically showing a schematic configuration in an example of the gene testing apparatus of the present disclosure. As shown in FIG. 1, the gene testing apparatus 1 includes a specimen container installation unit 2, a leukocyte separation unit 3, a concentration measurement unit 4, a reagent mixing unit 5, a second liquid treatment unit 6, an optical measurement unit 7, and a measurement processing unit 8. Note that these units are not necessarily configured as an integrated device, and parts that are appropriately separated as long as the units are arranged in proximity or electrically connected to form one device as a whole are also acceptable.

[0017] The gene to be tested in the gene testing apparatus 1 targets the gene possessed by leukocytes contained in blood. Similar to a general blood test, blood as a specimen is collected in advance, and the blood is contained in a specimen container. The specimen container installation unit 2 is a site for installing a specimen container containing blood obtained by blood collection as a specimen. As the specimen container, generally used blood collection tubes, blood collection bottles, sample cups, etc. are used. The specimen container installation unit 2 has no particular limitation on its configuration as long as it can easily install the specimen container, stably hold it, and reliably perform the operation of moving the blood to the site of the next process.

[0018] The leukocyte separation unit 3 is a site that collects blood from the specimen container of the specimen container installation unit 2, and then uses a leukocyte separation device 31 to separate leukocytes in the blood and recover a first liquid containing leukocytes as the main blood cells. The first liquid is a liquid containing leukocytes separated from the blood specimen as the main blood cell component, and is sometimes referred to as a leukocyte liquid hereinafter. Additionally, this first liquid is sometimes referred to as a leukocyte cell suspension in the HPA method.

[0019] In addition to the leukocyte separation device 31, the leukocyte separation unit 3 further includes a nozzle unit 32 having a syringe pump function for aspirating blood from the sample container and injecting it into the leukocyte separation device 31, and a moving drive unit 33 that moves the nozzle unit 32 from the sample container to the leukocyte separation device 31 for blood aspiration and injection.

[0020] The nozzle unit 32 draws blood from the sample container placed in the sample container placement unit 2, temporarily holds it, and then injects it into the leukocyte separation device 31 under appropriate conditions. First, the nozzle unit 32 moves to the sample container placement unit 2 and inserts its tip, such as a hypodermic needle, into the blood in the sample container. Next, it draws blood using the syringe pump function and temporarily holds it in the nozzle unit 32. Then, it moves to the leukocyte separation unit 3 and connects the tip of the nozzle unit 32 to a predetermined inlet port of the leukocyte separation device 31, and thereafter injects the held blood into the leukocyte separation device 31 under appropriate conditions. When injecting blood into the leukocyte separation device 31, the blood may also be introduced directly into the leukocyte separation device 31 from the nozzle unit 32 using the syringe pump function. Alternatively, an introduction mechanism having a cylinder portion with a predetermined volume and a movable packing may be placed in the introduction port of the leukocyte separation device 31, and after injecting blood into the cylinder portion, the packing may be pressed while maintaining an airtight state with a plunger fitted to it, thereby introducing the blood into the leukocyte separation device 31 under predetermined appropriate introduction conditions.

[0021] The mobile drive unit 33 is a mechanism that moves the nozzle unit 32 between the sample container and the leukocyte separation device 31 as described above. The mobile drive unit 33 also uses the syringe pump function of the nozzle unit 32 to cause the nozzle unit 32 to aspirate blood from the sample container and the nozzle unit 32 to move between the sample container and the leukocyte separation device 31. This is a mechanism that drives the injection of blood. Such a mobile drive unit 33 can be used in combination with a mobile mechanism such as various automated arms or robot arms and a drive mechanism such as a gear type, hydraulic type, water pressure type, wire type or electric type.

[0022] Furthermore, the leukocyte separation unit 3 may have a nozzle cleaning unit so that the nozzle unit 32 can be easily reused. The nozzle cleaning unit may simply be a cleaning container that holds a cleaning solution. A moving drive unit 33 can be used to move the nozzle unit 32 to the nozzle cleaning unit and perform the cleaning operation. This allows the nozzle unit 32 to be reused repeatedly, and the gene testing device 1 can be used efficiently.

[0023] Here, the leukocyte separation device 31 used in the gene testing apparatus 1 of this disclosure will be described with reference to the drawings. Figure 2 is a schematic plan view showing an example of the leukocyte separation device 31. The leukocyte separation device 31 is a device called a flow channel device, and in the following description, a "flow channel" has a structure through which liquid flows. The length of the flow channel in a direction perpendicular to the direction in which the flow channel extends is called the width of the flow channel. A relatively small width of the flow channel means that the flow channel is relatively narrow, and a relatively large width of the flow channel means that the flow channel is relatively wide.

[0024] The leukocyte separation device 31 has a plate-shaped base. The leukocyte separation device 31 has a surface (also called the first upper surface) 31a, a surface opposite to the first upper surface 31a (also called the first lower surface) 31b, and a surface (also called the first side surface) 31c that connects the first upper surface 31a and the first lower surface 31b. The outer surface of the leukocyte separation device 31 is composed of the first upper surface 31a, the first lower surface 31b, and the first side surface 31c. The first upper surface 31a is located on the +Z side of the first lower surface 31b.

[0025] In the example shown in Figure 1, the first upper surface 31a is oriented in the +Z direction. The first upper surface 31a has a normal vector along the +Z direction. The first lower surface 31b is oriented in the -Z direction. The first lower surface 31b has a normal vector along the -Z direction. Both the first upper surface 31a and the first lower surface 31b are basically flat and have a rectangular shape.

[0026] The thickness (length along the +Z direction) of the leukocyte separation device 31 is, for example, about 1 millimeter (mm) to 5 mm. The width (length along the +X direction) of the first upper surface 31a and the first lower surface 31b of the leukocyte separation device 31 is, for example, about 10 mm to 50 mm. The length (length along the +Y direction) of the first upper surface 31a and the first lower surface 31b of the leukocyte separation device 31 is, for example, about 10 mm to 30 mm.

[0027] The leukocyte separation device 31 includes a flow channel 330 on the outer surface of the substrate of the leukocyte separation device 31 that is not open, and a plurality of holes 332 that are connected to the flow channel 330 so that a fluid such as liquid can flow through them, and which are open on the outer surface of the substrate of the leukocyte separation device 31. In other words, the flow channel 330 is located inside the leukocyte separation device 31, and the flow channel 330 does not open to either the first upper surface 31a or the first lower surface 31b.

[0028] Figure 3 is a schematic plan view showing an example of the configuration of the flow channel section 330 and multiple holes 332 in the leukocyte separation device 31. In Figure 3, the flow channel section 330, two inlet holes 3325, 3327 and three The outer edges of the discharge holes 3326, 3328, and 3329 are drawn with solid lines. Figure 4 shows a portion of the flow channel 330 in region IV, which is enclosed by a dotted rectangle in Figure 3. In Figure 4, the outer edges of the main flow channel 334, the multiple branching flow channels 331, and the two flow channels 335 and 337 are drawn with solid lines.

[0029] The flow channel section 330 has a cross-sectional shape that is not open on the outer surface of the substrate of the leukocyte separation device 31. For example, it has a configuration in which multiple rectangular flow channels are connected. The flow channel section 330 is, for example, the first flow A channel (also called the main channel) 334 and multiple channels (branched channels) as multiple secondary channels. (Also called road) Includes 331.

[0030] The main flow path 334 is, for example, a straight flow path extending along the -Y direction as the first direction. Yes. The main channel 334 has an upstream section (also called the first upstream section) 3341 and a downstream section (also called the first downstream section) (u) It has 3342. The main channel 334 flows from the first upstream section 3341 toward the first downstream section 3342. It extends in the -Y direction.

[0031] Each of the multiple branch channels 331 is connected to the side of the main channel 334, and the main channel 334 It is also very narrow. Each of the multiple branch channels 331 opens on the side in the second direction, +X, which is perpendicular to the first direction, -Y, between the first upstream section 3341 and the first downstream section 3342 of the main channel 334. The main channel 334 has multiple sections (also called connection sections) C1 to which each of the multiple branch channels 331 is connected. Each of the multiple branch channels 331 is They branch off from the main channel 334 at different positions in the -Y direction, which is one direction. Multiple branch channels 331 are connected to multiple connection points C1, which are considered as the first direction - They are located at different positions in the Y direction.

[0032] In the examples in Figures 2 and 3, each of the multiple branching channels 331 is in the +X direction as the second direction. They extend along the direction and are aligned along the -Y direction, which is the first direction. Here, the multiple branch channels 331 constitute a group of branch channels 331 (also called a branch channel group) 331g. The number of branch channels 331 can be set to, for example, several tens to several hundred. (Figures 2 and 3) For convenience, 13 branch channels 331 are depicted.

[0033] The multiple holes 332 include an introduction hole 3325 as a first introduction hole and an introduction hole 3327 as a second introduction hole. It includes a discharge hole 3329 as a first discharge hole, a discharge hole 3326 as a second discharge hole, and a discharge hole 3328 as a third discharge hole.

[0034] The inlet 3327 is a second inlet, serving as a pressure liquid inlet for introducing the pressure liquid, and is connected to the first upstream section 3341 of the main flow path 334 via the flow path 337. In this example, the inlet 3327 opens to the first upper surface 31a. The flow path section 330 consists of the inlet 3327 as the first inlet and the first It includes a channel 337 as a third channel connecting to the upstream section 3341. Channel 337 is wider than each branch channel 331. The diameter of the inlet hole 3327 is set to be equal to or greater than the width of channel 337. In this example, the flow path 337 has an opening on the side opposite to the second direction, the +X direction, of the main flow path 334, where it is connected to the first upstream section 3341. In the examples of Figures 2 and 3, the flow path 337 has a portion that extends from the inlet hole 3327 along the first direction, the -Y direction, and the second The L-shaped channel extends along the +X direction, opposite to the two directions, and is connected to the first upstream section 3341, in the order described above. The channel 337 is in the -Y direction and the +X direction. They extend in the direction indicated.

[0035] The inlet 3325 is the first inlet, serving as a blood inlet for introducing blood, and the flow path 335 It is connected to the first upstream section 3341 of the main channel 334 via the inlet hole 3325 in this example. It is open to surface 31a. The flow channel section 330 has an inlet hole 3325 as the first inlet hole and a first upstream section 3341 It includes a channel 335 as a fourth channel connecting to the other channels. Channel 335 is thicker than each branch channel 331. The diameter of the inlet hole 3325 is set to be equal to or greater than the width of the flow path 335. In this example, the portion of the flow path 335 connected to the first upstream section 3341 is aligned along the -Y direction as the first direction. It extends in this direction. In the examples of Figures 2 and 4, the channel 335 is connected to the first upstream portion 3341 of the main channel 334 in the -Y direction as the first direction. More specifically, for example, the channel 335 has a portion that extends from the inlet hole 3325 along the -X direction opposite to the second direction, and the second The L-shaped channel is connected in the order described, with a portion extending along the -Y direction as one direction. The channel 335 extends in the order of -X direction and -Y direction.

[0036] The discharge port 3329 is a first discharge port serving as a leukocyte discharge port for discharging a fluid mainly containing leukocytes separated from blood, and is connected to the first downstream section 3342 of the main channel 334 via the channel 339. In this example, the discharge port 3329 opens to the first lower surface 31b. The channel section 330 is the first discharge port Includes a fifth flow path 339 that connects the discharge port 3329 as a discharge port 3329 to the first downstream section 3342. The flow path 339 is wider than each branch flow path 331. The diameter of the discharge hole 3329 is equal to or greater than the width of the flow path 339. The width is set to be greater than or equal to the width of the flow path 339. In this example, the portion of the flow path 339 connected to the first downstream section 3342 is open on the side surface of the first downstream section 3342 in the second direction, the +X direction. In the examples of Figures 2 and 3, the flow path 339 is connected to the first downstream section 3342 and in the second direction The U-shaped channel is connected in the order described above, with a portion extending along the +X direction as the first direction, a portion extending along the -Y direction as the first direction, and a portion extending along the -X direction opposite to the second direction. The channel 339 extends in the order of +X direction, -Y direction and -X direction. It is growing.

[0037] The discharge port 3326 is a second discharge port serving as a red blood cell discharge port for discharging a fluid mainly containing red blood cells separated from the blood, and is connected via the flow path 336 to the portion 3312 opposite to the main flow path 334 in each of the multiple branched flow paths 331 (also called the second downstream portion). The discharge port 3326 is, In this example, it opens to the first lower surface 31b. The flow channel section 330 has a discharge hole 3326 as the second discharge hole and As a sixth channel connecting the second downstream section 3312 in each of the multiple branch channels 331 The flow path 336 is included. More specifically, each of the multiple branched flow paths 331 is connected to the flow path 336 at different positions in the -Y direction as the first direction. The flow path 336 is wider than each of the branched flow paths 331. The diameter of the discharge hole 3326 is equal to or greater than the width of the flow path 336. It is set. In the example in Figures 2 and 3, the flow path 336 is an L-shaped flow path in which multiple second downstream sections 3312 of multiple branch flow paths 331 are connected to each other, and a portion extending linearly along the -Y direction as the first direction and a portion extending linearly along the +X direction as the second direction are connected in the order described. The flow path 336 is in the -Y direction and the +X direction They extend in sequence.

[0038] The discharge port 3328 is a third discharge port serving as a residual fluid discharge port for discharging the fluid containing the residual corrective material remaining after most of the red and white blood cells have been separated from the blood, and is connected to the first downstream section 3342 of the main flow channel 334 via the flow channel 338. In this example, the discharge port 3328 opens to the first lower surface 31b. The flow channel section 330 connects the discharge port 3328, which serves as the third discharge port, to the first downstream section 3342. It includes a channel 338 as the seventh channel. Channel 338 is wider than each branch channel 331. Discharge hole 3328 The diameter is set to be equal to or greater than the width of the channel 338. In this example, the channel 338 is The portion connected to the first downstream section 3342 extends along the -Y direction. In the examples of Figures 2 and 3, the channel 338 is connected to the first downstream section 3342 and the first direction is the -Y direction. The flow path is connected in the order described above, consisting of a portion extending along the -Y direction, a portion extending along the -X direction opposite to the second direction, a portion extending along the -Y direction as the first direction, and a portion extending along the +X direction as the second direction and connected to the discharge hole 3328. The flow path 338 extends in the order of -Y direction, -X direction, -Y direction, and +X direction.

[0039] In this example, each of the two inlet holes 3325 and 3327 opens to the first upper surface 31a, and each of the three discharge holes 3326, 3328, and 3329 opens to the first lower surface 31b. Inlet hole 3327 has a portion that opens to the first upper surface 31a (also called the first inlet or first entrance). Inlet hole 3325 has a portion that opens to the first upper surface 31a (also called the second inlet or second entrance). Discharge hole 3329 has a portion that opens to the first lower surface 31b (also called the first discharge port or first exit). Discharge hole 3326 has a portion that opens to the first lower surface 31b (also called the second discharge port or second exit). Discharge hole 3328 has a portion that opens to the first lower surface 31b (also called the third discharge port or third exit).

[0040] The function of the leukocyte separation device 31 can be broadly described as follows: A liquid containing multiple types of particles P100 and P200 (see Figure 5) (also called the liquid to be processed) is introduced into chair 31. The leukocyte separation device 31 separates the target particle P100, which is a specific type of particle, from others. The particles of this species (also called other species of particles) P200 are separated and discharged. In this example, the target of separation is Particle P100 is a white blood cell, and the other particle P200 is a red blood cell; the liquid being processed is blood. In other words, the white blood cell separation device 31 separates the white blood cells, which are the target particle P100, from the blood being introduced. The spheres are separated from other types of particles, such as red blood cells (P200), and then expelled. Note that there are three or more types of particles. This is also acceptable. Below, an example is given where the target particle P100 and the other particle P200 are each of the same type of particle.

[0041] The leukocyte separation device 31 receives a pressing fluid through the second introduction port, introduction port 3327. Additionally, the blood to be processed is received through the first introduction port, introduction port 3325. Specific examples and functions of the pressing fluid will be described later.

[0042] When the pressing fluid is introduced into the leukocyte separation device 31 from the introduction port 3327, in this example, the pressing fluid supply unit of the nozzle unit 32 may be connected to the introduction port 3327. The nozzle unit 32 may be directly connected to the introduction port 3327. Alternatively, in order to connect the nozzle unit 32, there may be a cylindrical portion on the first upper surface 31a of the leukocyte separation device 31 that surrounds the introduction port 3327 around the Z axis when viewed in plan (hereinafter, in plan view taken in the -Z direction unless otherwise specified) and protrudes in the +Z direction.

[0043] When blood is introduced into the leukocyte separation device 31 from the introduction port 3325, in this example, the blood aspiration / injection portion of the nozzle portion 32 may be connected to the introduction port 3325. The nozzle portion 32 may be directly connected to the introduction port 3325. Alternatively, in order to connect the nozzle portion 32, there may be a cylindrical portion on the first upper surface 31a of the leukocyte separation device 31 that, when viewed from above, surrounds the introduction port 3325 around the Z axis and protrudes in the +Z direction.

[0044] The blood introduced into the leukocyte separation device 31 from the introduction port 3325 passes through the channel 335 and then flows through the main channel. It flows into the first upstream section 3341 of channel 334. It is introduced into the leukocyte separation device 31 through the introduction port 3327. The pressing liquid flows into the first upstream section 3341 of the main channel 334 via the channel 337.

[0045] In Figure 4, the dotted arrow Fp1 indicates the direction in which the pressing liquid is directed. This direction is along the +X direction. In Figure 4, the thicker dotted arrow Fm1 indicates the direction in which the main flow (also called the main stream) of the liquid to be treated flows from the channel 335 into the main channel 334. This main stream is directed along the -Y direction, which is the first direction. In Figure 4, the thin dotted rectangle virtually represents the outer edge of the first upstream section 3341.

[0046] Figure 4 schematically shows how the particles P100 to be separated are separated from each other when their diameter is larger than that of other particles P200. Specifically, the width of each branch channel 331 is The diameter is larger than that of red blood cells, which are other particle types P200, and smaller than that of white blood cells, which are the target particle P100 for separation. Here, the width of the branched channel 331 is the length of the branched channel 331 along the Y direction.

[0047] At least the width of the main channel 334 and the channel 335 respectively is such that the target particles P100 and other types It is larger than any of the diameters of particle P200. Here, the width of the main channel 334 is the length of the main channel 334 along the X direction which is perpendicular to the Y direction as the first direction. The width of channel 335 is near the main channel 334. This is the length of the channel 335 along the X direction next to it. The width of the channel 335 is the width of the channel 335 along the -X direction At the point where it extends along the Y-axis, it is the length of the flow path 335 along the Y-axis.

[0048] Other particles P200 move in the main channel 334 in the -Y direction as the first direction, and are subjected to a force pushing them in the +X direction, so most of them are guided into one of the multiple branch channels 331. Most of the other particles P200 pass through one of the multiple branched channels 331 and then through channel 336 to be discharged outside the leukocyte separation device 31 through the discharge port 3326. By adjusting the cross-sectional area and length of each branch channel 331 connected to the main channel 334, other particles P200 are introduced from the main channel 334 into one of the branch channels 331 and separated from the target particles P100. Other particles P200 discharged to the outside of the leukocyte separation device 31 from the discharge port 3326 are, for example, For example, other particles P200 discharged from the discharge port 3326 to the outside of the leukocyte separation device 31 may be subjected to specific processing in other devices connected directly to the discharge port 3326 or via other components such as tubes, or they may simply be collected in a container. It may be discarded via other components.

[0049] The target particle P100 is hardly introduced into the multiple branch channels 331, and instead passes through the main channel 334. It moves in the -Y direction as one direction. Most of the target particles P100 are discharged outside the leukocyte separation device 31 through the main channel 334 and then through the channel 339 and discharge port 3329. Here, the width of the channel 339 is greater than the width of the particle P100 to be separated. Similar to how seed particles P200 are introduced into one of the multiple branched channels 331, the particles P100 to be separated that reach the first downstream section 3342 flow into channel 339 instead of channel 338. The particles P100 to be separated that are discharged from the discharge port 3329 to the outside of the leukocyte separation device 31 may be subjected to specific processing in another device connected to the discharge port 3329 directly or via other components such as a tube, or they may simply be collected in a container. In this example, they are used as the target of measurement in the following concentration measurement section 4.

[0050] In this example, the flow (also called the introduction flow) introduces the blood, which is the liquid to be processed, into the branch channel 331. The following is used. The introduction flow separates the target particles P100 through the main flow channel 334 and multiple branched flow channels 331. This can contribute to the separation of other particles P200. The introduction flow is shown in Figure 4, using a hatch made of sand. The region Ar1 is indicated by the ring mark. The introduction flow shown by region Ar1 in Figure 4 is merely an example and can change depending on the relationship between the flow velocity and flow rate of the liquid to be treated introduced from channel 335 to the main channel 334 and the flow velocity and flow rate of the pressing liquid introduced from channel 337 to the first upstream section 3341 of the main channel 334. Region Ar1 is adjusted as appropriate so that the particles to be separated P100 and other particles P200 are efficiently separated from the liquid to be treated. The pressing liquid presses the liquid to be treated into the multiple branch channels 331 in the +X direction from the opposite side of the multiple branch channels 331. The pressing liquid can contribute to the generation of the introduction flow.

[0051] Here, as described above, the main flow path 334 extends in the -Y direction, which is the first direction. The portion of the flow path 335 connected to the first upstream section 3341 of the main flow path 334 extends along the -Y direction, which is the first direction. Each of the multiple branch flow paths 331 opens on the side in the +X direction, which is the second direction, between the first upstream section 3341 and the first downstream section 3342 of the main flow path 334. Flow path 337 opens on the side in the -X direction opposite to the second direction of the first upstream section 3341 of the main flow path 334. Therefore, the pressing liquid is supplied to the main flow path 334 through the inlet hole 3327. Furthermore, by supplying the liquid to be treated (blood) containing multiple types of particles (white blood cells and red blood cells) to the main channel 334 via the introduction hole 3325, the multiple types of particles are processed in the main channel 334 through multiple branch channels 331. A liquid flow that pushes toward can be generated. This makes it easier for other types of particles P200, which are particles of a smaller diameter than the width of each branch channel 331 among the multiple types of particles, to flow into the multiple branch channels 331. As a result, for example, among the multiple types of particles in the liquid being treated, each This facilitates the separation of target particles P100 (white blood cells), which have a diameter larger than the width of the branch channel 331, from other particle types P200 (red blood cells), which have a diameter smaller than the width of each branch channel 331.

[0052] Furthermore, in this example, the portion of the channel 339 connected to the first downstream section 3342 of the main channel 334 is open on the side surface in the +X direction, which is the second direction of the first downstream section 3342. Therefore, for example, due to the action of the introduction flow in the main channel 334, separation target particles P100 with a diameter larger than the width of each branch channel 331 are more likely to flow into the channel 339. As a result, the separation target particles P100 (white Blood cells can be easily discharged from the first channel device 31 through the channel 339 and the discharge port 3329. As a result, for example, among the multiple types of particles in the liquid to be treated, the width of each branch channel 331 This facilitates the separation of the target particle P100, which has a larger diameter, from other particle species P200, which has a diameter smaller than the width of each branch channel 331.

[0053] In Figure 4, the width of the inlet flow in the main channel 334 branches out from the main channel 334 into multiple branch channels 331. It is shown as width W1 near the region where it is introduced. The flow width is the length of the introduction flow along the X direction. The width W1 is, for example, the main flow path 334 and the secondary flow path. Adjustment of the cross-sectional area and length of each of the branched flow channels 331 and the liquid to be processed and the liquid to be pressed It can be set by adjusting the flow rate.

[0054] In Figure 4, the width W1 is such that the centroid of the target particle P100 is not included in the introduction flow region Ar1. The example is given with a width that includes the center of gravity of other particle types, such as P200.

[0055] In this example, blood is used as the liquid to be processed. In this case, the target particle P100 These are white blood cells, and the other particle P200 is a red blood cell. In this example, the specific treatment for the target particle P100 involves measuring the concentration (or number) of white blood cells in the concentration measurement unit 4, followed by reagent mixing, reaction processing, and detection of chemiluminescence. It flows through the channel 338. An example of the residual composition discharged from the leukocyte separation device 31 through the discharge port 3328 is plasma. In this case, phosphate-buffered saline (PBS) is used as an example of the pressing fluid. In order to give the pressing fluid a function according to the intended use of the leukocyte separation device 31, a liquid in which other components are added to PBS may be used as the pressing fluid.

[0056] The center of gravity of a red blood cell is located approximately 2 to 2.5 micrometers (μm) from the outer edge of the red blood cell. The position is such that the maximum diameter of a red blood cell is approximately 6 μm to 8 μm. The center of gravity of a white blood cell is approximately 5 μm to 10 μm from the outer edge of the white blood cell. The maximum diameter of a white blood cell is approximately 10 μm to 30 μm. From the perspective of separating red blood cells and white blood cells in the blood, a value of approximately 2 μm to 15 μm is adopted for the width W1 of the introduction flow.

[0057] The cross-sectional area of ​​the virtual cross-section of the main channel 334 along the XZ plane is, for example, 300 square micrometers (μm). 2 ) to 1000 μm 2 It is to that extent. The length of the main channel 334 along the Y direction is, for example, The thickness ranges from approximately 0.5 mm to 20 mm. The cross-sectional area of ​​the hypothetical cross-section of the branch channel 331 along the YZ plane is, for example, 100 μm². 2 From 500 μm 2 It is approximately as follows. The length of the branch channel 331 along the X direction is, for example, For example, it is about 3 mm to 25 mm. In the main flow path 334, the direction is in the -Y direction as the first direction. The flow velocity of the liquid being treated is, for example, between 0.2 meters per second (m / s) and approximately 5 m / s. Furthermore, the liquid flow rate per unit time in the main channel 334 is, for example, approximately 0.1 microliters per second (μl / s) to 5 μl / s.

[0058] In the leukocyte separation device 31, the total volume of the flow channel 330, the two inlet ports 3325, 3327 and the three outlet ports 3326, 3328, 3329 can be set to approximately 0.5 microliters (μl) to 2 μl. The total volume of the main flow channel 334, the two flow channels 335, 337 and the two inlet ports 3325, 3327 is The volume can be set to approximately 0.07 μl to 0.3 μl. The total volume of the main channel 334, the four channels 335, 337, 338, 339, the two inlet holes 3325, 3327, and the two outlet holes 3328, 3329 can be set to approximately 0.1 μl to 0.5 μl.

[0059] For example, a resin such as polydimethylsiloxane (PDMS) is used as the material for forming the leukocyte separation device 31. PDMS has excellent transferability when performing resin molding using a mold. Transferability is the property of forming fine irregularities in the resin molded product according to the fine pattern of the mold.

[0060] The leukocyte separation device 31 has, for example, fine irregularities on one side corresponding to the pattern of the flow channel section 330. The first part can be manufactured by joining a plate-shaped first part and a plate-shaped second part having five through holes corresponding to two inlet holes 3325, 3327 and three outlet holes 3326, 3328, 3329, such that one side of the second part covers the fine irregularities of the first part. The first part having fine irregularities on one side can be manufactured by resin molding or the like. The second part having five through holes may be manufactured by resin molding, or by forming five through holes in a flat plate-shaped member formed by resin molding using a punching process or the like. The joining of the first part and the second part can be achieved without using adhesive, for example, by surface modification of one side of the first part and one side of the second part, and by contact between one side of the first part and one side of the second part. Surface modification can be achieved, for example, by irradiation with oxygen plasma or irradiation with ultraviolet (UV) light using an excimer lamp. If one side of the first part and one side of the second part are made of the same type of resin, the strength of the bond between one side of the first part and one side of the second part using surface modification may be improved.

[0061] The gene testing apparatus 1 of this disclosure includes a leukocyte separation unit 3 having such a leukocyte separation device 31. Blood is introduced into the leukocyte separation device 31 through a nozzle 32 via a first introduction port 3325 and a pressing fluid is introduced through a second introduction port 3327. This allows the blood cells in the blood to be separated into leukocytes that pass through the main channel 334 and red blood cells that are introduced into multiple branch channels 331. As a result, the leukocytes that pass through the main channel 334 are identified as the main blood cells. A first solution, which is a leukocyte solution containing globules, can be generated and collected by discharging it from the first discharge port 3329, and the first solution can be used in subsequent testing steps.

[0062] The first liquid obtained by the leukocyte separation unit 3 is then sent to the concentration measurement unit 4, which is equipped with a first light incidence means and a first light detection means. To send the first liquid from the leukocyte separation unit 3 to the concentration measurement unit 4, the collection container, which has been connected to the first discharge port 3329 of the leukocyte separation device 31 and collected the first liquid, may be moved to the measurement position of the concentration measurement unit 4 by a moving means similar to the moving drive unit 33 and used for measurement. In this case, it is preferable to use a translucent container for the collection container in order to optically measure the concentration of leukocytes. Alternatively, the first liquid may be sent to a measurement container placed at the measurement position of the concentration measurement unit 4 via a tube or the like connected to the first discharge port 3329. The measurement container should basically be a translucent container suitable for optical measurement. For the movement of the first liquid, various known mechanisms can be applied, as long as they allow for optical concentration measurement of the collected first liquid after movement.

[0063] In the concentration measurement unit 4, a first light incidence means is used to inject light for measurement into the first liquid, and a first light detection means is used to detect reflected light from the first liquid or transmitted light that has passed through the first liquid. The first light incidence means and the first light detection means may each be composed of separate, independent elements arranged in a manner suitable for detecting reflected light or transmitted light. Alternatively, a light-emitting element as the first light incidence means and a light-receiving element as the first light detection means may be integrally formed on a single semiconductor substrate in a manner suitable for detecting reflected light, forming a light-receiving sensor. Various known methods for calculating the concentration of the target to be detected from the detection result of the first light detection means can be applied to the optical concentration measurement of leukocytes in the first liquid using the first light incidence means and the first light detection means.

[0064] Next, we will explain in more detail the optical inspection method for specific regions of genes using the HPA method described above. Here, we will describe a method for measuring the length of the G-tail sequence, which is the length of the G-tail 92 of telomere 91 on chromosome 90 of the gene.

[0065] In measuring the length of G-tail 92, an AE (acridinium ester) labeled probe is used as the probe reagent. Here, an acridinium ester refers to an acridinium ring and an ester group, for example, 4-(2-succinimidyloxycarbonylethyl)phenyl-10-methylacridinium 9, a compound having a phenyl ester group. -This refers to carboxylates. A specific compound name is dimethylacridinium ester (DMAE). G-tail 92 is located at the end of the G-chain of the telomere duplex portion consisting of the G-chain and C-chain of telomere 91 at the end of chromosome 90.

[0066] The AE-labeled probe used as a probe reagent is labeled with AE and has a sequence complementary to the repeat sequence in G-tail 92. Therefore, a number of probes corresponding to the number of repeats in the repeat sequence hybridize to G-tail 92 through hybridization (the complementary formation of complexes by nucleic acid molecules; also called molecular hybridization). Specifically, hybridization between the AE-labeled probe and G-tail 92 can be performed by adding a hybridization solution containing the AE-labeled probe to a cell pellet and incubating it at, for example, 60-65°C for 5-30 minutes. The AE-labeled probe hybridized with G-tail 92 is stabilized, and the ester bonds of the AE are protected even after hydrolysis for a certain period of time. Therefore, the AE can chemiluminescent when hydrogen peroxide and alkaline solutions are added. The length of G-tail 92 can be measured by detecting the amount of luminescence with a photodetector and quantitatively calculating from the detection result.

[0067] On the other hand, in probes where the probe and G-tail 92 do not hybridize, the AE is not stabilized. If hydrolysis is performed in this state, the ester bond of the AE undergoes hydrolysis, resulting in a deactivated probe, and no chemiluminescence occurs at all, so the deactivated probe will not be detected. To exclude chemiluminescence based on unreacted probes, hydrolysis can be specifically performed by adding a hydrolysis reagent and incubating at 60°C for 5 to 10 minutes. The amount of chemiluminescence of the AE after incubation can be measured using an appropriate second photodetector, such as a luminometer.

[0068] The basic method is as described above, but further treatment involves treating the DNA sample with exonuclease I (ExoI) to selectively remove single-stranded nucleotides and then selecting the G-tail sequence. It is preferable to selectively remove the contaminants and confirm that the chemiluminescence is specific to the G-tail 92. Alternatively, the ratio of the detection result from a sample not treated with ExoI to the detection result from a sample treated with ExoI can be calculated as the signal-to-noise ratio (S / N). This allows for the specific measurement of the G-tail 92 length even if contaminants are present in the sample. Furthermore, treating the sample with T7 exonuclease to remove the C-chain of telomere 91 and increase the G-tail 92 of the G-chain of telomere 91 also confirms that the chemiluminescence is specific to the G-tail 92 sequence.

[0069] In the method described above, for example, assuming that the basic unit sequence of the G-tail 92 repeat sequence (5'-TTAGGG-3') is repeated 24 times, and that a probe consisting of four repeats of the sequence (5'-CCCTAA-3') (5'-(CCCTAA)4-3') is used, then theoretically, six probes can be hybridized into the G-tail 92. Therefore, in this case, labels equivalent to six probes will be detected. If the intensity of the labels detected when such probes are hybridized into DNA standards of known length is determined in advance, and a calibration curve is created based on this, the length of the G-tail 92 can be calculated by converting it to the length of the G-tail sequence.

[0070] Furthermore, when acridine derivatives other than AE, or other non-radioactive labeling substances (e.g., luminol, isolminol, pyrogallol, protohemin, aminobutylethyl-n-isoluminol, aminohexylethyl-n-ethyl-isoluminol) are used as labels, the length of the G-tail 92 can be measured in the same way by quantifying the amount of chemiluminescence. Even when these labels are used, an appropriate amount of probe labeled with an acridine derivative or other non-radioactive labeling substance is added to the cell pellet and reacted. After the reaction is complete, hydrolysis or other treatments are performed, and then chemiluminescence is observed, the amount of luminescence is detected and calculated for quantification.

[0071] In the gene testing apparatus 1 of this disclosure, the concentration of leukocytes in the first solution is optically measured in the concentration measurement unit 4. Then, in the reagent mixing unit 5, the cell membrane lysate is mixed with the first solution, the temperature is adjusted, and then the probe reagent is mixed to prepare the second solution, which is a mixture of the first solution and the probe reagent. In this first solution, the cell membrane of the leukocytes is lysed by mixing with the cell membrane lysate, and the genes (DNA) that the leukocytes possessed are extracted into the solution. To send the first solution from the concentration measurement unit 4 to the reagent mixing unit 5, a recovery container or measuring container placed in the concentration measurement unit 4 may be moved from the measurement position of the concentration measurement unit 4 to the processing position of the reagent mixing unit 5 using a moving means similar to that of the moving drive unit 33 and used for mixing the reagents. Alternatively, the first solution may be sent using a mechanism similar to that of the nozzle unit 32, which can aspirate the first solution from the recovery container or measuring container placed in the concentration measurement unit 4, move it to the processing position of the reagent mixing unit 5, and introduce it into the mixing container.

[0072] In preparing the second solution, it is preferable to extract DNA, which is chromosome 90, from the nucleus of leukocytes and react a specific site of the gene possessed by the leukocyte with the probe reagent by lysing the cell membrane of the leukocytes and extracting the DNA, which is the gene possessed by the leukocytes, into the solvent and suspending it. Various known methods can be applied to extract DNA from leukocytes. For example, a method can be applied in which a cell membrane lysis solution, which is a buffer for lysing cell membranes, nuclear membranes, etc., is added to the first solution to lyse the cell membrane of the leukocytes and extract DNA from the leukocytes. Examples of such cell membrane lysis solutions include lithium succinate buffer containing lauryl sulfate, lithium chloride, EDTA (ethylenediaminetetraacetic acid), and EGTA (ethylene glycoltetraacetic acid (also called glycol etherdiaminetetraacetic acid)).

[0073] By mixing the cell membrane lysate with the first solution, the cell membranes of leukocytes are lysed and the gene (chromosome 90) is extracted without any special operations such as heating. Then, by adjusting the temperature of the solution (first solution mixed with cell membrane lysate), for example, by maintaining it at room temperature, the gene (chromosome 90) that was retained in the cell membrane is extracted. In this state, the gene has telomeres 91, and G-tails 92 are present at its ends, as shown in Figure 5. After adjusting the temperature in this way, by mixing in the probe reagent, the labeled probe in the probe reagent can react with the G-tail 92, a specific site in the single-stranded state.

[0074] Furthermore, by adjusting the temperature of the solution obtained by mixing the first solution with the cell membrane lysate, for example, by heating it to 90-95°C, the double strands of DNA at telomere 91 can be separated after the leukocyte cell membrane has lysed, resulting in a state where a single strand with a length corresponding to the telomere length exists. After separating the double strands, the solution is rapidly cooled, for example by ice cooling, to lower the temperature of the liquid to room temperature. By mixing the probe reagent into this first solution, the labeled probe in the probe reagent can be reacted with telomere 91, a specific region in the single-strand state, according to the telomere length.

[0075] As described above, a second solution for reacting the probe reagent with a specific site of the gene can be prepared in the reagent mixing unit 5. To efficiently carry out the above process, the reagent mixing unit 5 preferably includes, for example, a lysis solution mixing means for mixing the cell membrane lysis solution with the first solution, a temperature adjustment means for heating and cooling the mixed solution as needed after mixing the cell membrane lysis solution with the first solution, and a reagent mixing means for mixing the probe reagent. Each of these means may be the same as those described above or later, or various known means.

[0076] The second solution, prepared by mixing the cell membrane lysis solution and probe reagent with the first solution in the reagent mixing unit 5, is sent to the second solution processing unit 6. The second solution processing unit 6 is preferably located close to the reagent mixing unit 5 in order to perform processing using the container used for mixing in the reagent mixing unit 5. The movement of the container from the reagent mixing unit 5 to the second solution processing unit 6 is, for example, driven by the movement drive unit 33. The reagent can be moved from the processing position in the reagent mixing unit 5 to the processing position in the second liquid processing unit 6 using a similar means of transport.

[0077] In the second solution processing unit 6, the second solution is heated and cooled to react specific sites of genes in the leukocytes in the second solution with the probe reagent, thereby forming a luminescent region at the specific site of the genes in the leukocytes in the second solution. Here, first, the second solution is heated. The second solution, which contains DNA extracted from leukocytes, is then cooled to, for example, 60-65°C. As a result, since the probe reagent is mixed into the second solution, the labeled probe hybridizes to the single-stranded portion of the DNA, such as the G-tail 92 portion or the telomere 91 portion, which is in a single-stranded state. The reaction time is, for example, about 5-20 minutes. As a result, a luminescent region is formed by the labeled probe at the specific site of the DNA, which is the gene in the leukocytes in the second solution.

[0078] Therefore, the reaction between the specific site of the leukocyte in the second solution and the probe reagent at this time is, more specifically, a reaction in which the labeled probe of the probe reagent hybridizes with the G-tail 92 or telomere 91 of the DNA extracted by lysing the leukocyte. The labeled probe hybridized with the G-tail 92 or telomere 91 forms a luminescent part, and this luminescent part chemiluminescents through the next process.

[0079] As the labeled probe used in the probe reagent, the aforementioned AE-labeled probe can be used. Alternatively, for example, an oligonucleotide having a base sequence represented by (CCCTAA)n (where n is an integer from 1 to 10) and labeled with at least one non-radioactive labeling substance can be used. This n can be appropriately selected depending on the target DNA. For example, n is preferably 2 to 8, and more preferably 3 to 5. The oligonucleotide used in the labeled probe can be manufactured using a commercially available DNA synthesizer by any DNA manufacturing method, such as the phosphoamidite method. It is preferable to introduce, for example, an aminolinker for labeling with a non-radioactive labeling substance during this chemical synthesis.

[0080] AE can label oligonucleotides to which aminolinkers have been introduced by the reaction of the amino group of the introduced aminolinker with the N-hydroxysuccinimide ester of AE. This allows for the construction of labeled probes for use in the gene testing apparatus of this disclosure. The labeling position of AE, etc., can be freely set by the position of the aminolinker introduced during DNA synthesis.

[0081] Examples of non-radioactive labeling substances include luminol, isolminol, pyrogallol, protohemin, aminobutylethyl-n-isoluminol, or aminohexylethyl-n-ethyl-isoluminol, in addition to AE. Non-radioactive labeling substances have substituents that can chemically bond with the amino group of the aminolinker introduced into the oligonucleotide. Examples of such substituents include the N-hydroxysuccinimide ester group.

[0082] As a result, the second solution processing unit 6 forms a luminescent region at a specific site of the gene present in the leukocytes in the second solution. In this process, to remove chemiluminescence based on unreacted labeled probes, for example, a hydrolysis reagent can be added to the second solution and incubated at 60°C for 5-10 minutes to inactivate any unreacted, free probe-labeled substances by hydrolysis. Examples of hydrolysis reagents include tetraboric acid containing Triton X-100. Sodium buffer can be used.

[0083] The second liquid processing unit 6 includes a processing container for holding the second liquid, a heating means 61 for heating the processing container, and a heat dissipation means for cooling the processing container, in order to perform the above processing on the second liquid. It is preferable to have at least one of the stage and the cooling means 62, and a temperature detection means 63 for detecting the temperature of the processing container.

[0084] The processing container for the second liquid may be the same container used for mixing in the reagent mixing unit 5. Alternatively, a separate container may be used as the processing container for transferring the second liquid from the container used for mixing in the reagent mixing unit 5.

[0085] The heating means 61 for heating the second liquid can be any known heating means, such as any type of heater, a fan that supplies hot air to cover the processing container, or a water heater that supplies hot water to immerse the processing container. As the heating means 61 in the second liquid processing unit 6, any configuration can be used as long as it can quickly and stably heat the second liquid contained in the processing container to, for example, 60°C or 95°C.

[0086] As at least one of the heat dissipation means and cooling means 62 for cooling the heated processing container, various known heat dissipation means and cooling means can be applied, as long as they can cool the second liquid contained in the processing container from, for example, 95°C to 60°C, or from 60°C to room temperature (room temperature, usually 20-25°C), or even lower to a temperature close to 0°C, which is called ice cooling. As heat dissipation means, for example, a jacket equipped with a cooler such as an air cooling mechanism or a water cooling mechanism that is in contact with the surroundings of the processing container, a cooler that supplies cold air to cover the processing container, or a cooler that supplies cold water to immerse the processing container can be applied. Furthermore, if a heating and cooling mechanism using a Peltier temperature control method is adopted, for example, heating and cooling can be easily switched by reversing the direction of the supplied current, so the mechanism of the device can be made compact as a temperature control means that combines heating and cooling means, which is preferable. Furthermore, since both heat dissipation means and cooling means often include a heat dissipation mechanism for releasing heat to the outside, it is preferable to position the heat dissipation mechanism at the end of the Ide inspection device 1 in order to efficiently dissipate heat in such cases.

[0087] The temperature detection means 63 for detecting the temperature of the processing container can employ various known means used for temperature detection or measurement, and is not particularly limited as long as it can detect or measure the temperature of the second liquid during processing, either through the processing container or directly. For example, various types such as thermistors and thermocouples can be used, and it is particularly preferable if the temperature detection result can be output as an electrical signal, as this allows for accurate and stable temperature control.

[0088] The second solution processed in the second solution processing unit 6 is sent to the optical measurement unit 7. The optical measurement unit 7 measures the amount of light emitted by causing the light-emitting portion formed by the labeled probe in the DNA in the second solution to chemiluminescence. Therefore, light shielding is required to prevent the intrusion of external light during measurement. For this reason, it is preferable to place the optical measurement unit 7 in the center of the gene testing device 1 in a manner that ensures both light shielding and good operability. Specifically, in the gene testing device 1, the sample container placement unit 2, the leukocyte separation unit 3, the concentration measurement unit 4, the reagent mixing unit 5, and the second solution processing unit 6 are arranged in this order from left to right, and it is preferable that the optical measurement unit 7 is positioned behind the concentration measurement unit 4 and the reagent mixing unit 5 and second solution processing unit 6. This arrangement is preferable because it allows for the efficient sequential execution of a series of processes from the placement of the sample container to the detection of the amount of light emitted by the optical measurement unit 7.

[0089] Note that the left-right direction here can also be defined as the first direction in the gene testing device 1, for example, the direction corresponding to the left-right direction in the block diagram of the schematic configuration of the gene testing device 1 shown in Figure 1. In other words, when the operator is facing the gene testing device 1, it is the direction aligned with the left-right direction from the operator's perspective. Also, the rear direction is perpendicular to the first direction in the gene testing device 1. It can also be described as one of the two intersecting directions, for example, in the block diagram of the schematic configuration of the gene testing device 1 shown in Figure 1, it corresponds to the direction towards the back in the front-to-back direction of the diagram. In other words, when the operator faces the gene testing device 1, it is the part located towards the back in the depth direction from the operator's perspective.

[0090] The container holding the second liquid processed in the second liquid processing unit 6 can be moved to the optical measurement unit 7 by means of a moving mechanism similar to that of the moving drive unit 33, for example, by moving it from the processing position in the second liquid processing unit 6 to the optical measurement position in the optical measurement unit 7. The container used when processing the second liquid in the second liquid processing unit 6 is preferably translucent in order to optically detect the amount of light emitted in the optical measurement unit 7, if it is to be moved to the optical measurement unit 7 as is. Alternatively, the second liquid processed in the second liquid processing unit 6 may be sent to the optical measurement unit 7 and then transferred to an optical measurement container having translucency and a shape suitable for detecting the amount of light emitted. Furthermore, such an optical measurement container may be used as a container for holding the first and second liquids from the time the concentration of white blood cells is measured in the concentration measurement unit 4 until the amount of light emitted is detected in the optical measurement unit 7.

[0091] In the optical measurement unit 7, hydrogen peroxide and an alkaline solution are mixed with a second solution containing a gene on which a light-emitting part has been formed, more specifically, DNA in which a labeled probe has been hybridized to the G-tail 92, to cause the light-emitting part to chemiluminescent. The amount of light emitted from the light-emitting part is then detected by a second photodetector.

[0092] The hydrogen peroxide solution and alkaline solution to be mixed with the second solution should be mixed sequentially with the second solution. Alternatively, these hydrogen peroxide solution and alkaline solution may be mixed as a pre-mixed alkaline hydrogen peroxide solution. These hydrogen peroxide solution and alkaline solution function as triggers for the chemiluminescence of the labeled probe. The roles of these liquids are as follows: hydrogen peroxide (H2O2) acts as an oxidizing agent, and the alkaline solution acts as an oxidation aid. Basically, by mixing the hydrogen peroxide solution and alkaline solution in this order, the chemiluminescence of the labeled probe starts quickly. The time from mixing these liquids to the start of chemiluminescence is, for example, about 0.5 seconds. The alkaline solution may contain, for example, sodium hydroxide (0.01~1N (normal), preferably about 0.25N or 0.25~2M (mol / l), preferably about 1M) A solution of NaOH can be used. As hydrogen peroxide solution, for example, a concentration of 0.001 to 5% can be used. Hydrogen peroxide solution containing 0.1 to 0.5% of a certain degree can be used.

[0093] It is preferable to use a pre-luminescence mixing means 72 to mix hydrogen peroxide solution and alkaline solution with the second liquid. As such a pre-luminescence mixing means 72, for example, a mechanism having a syringe pump function for drawing these liquids from a predetermined container and injecting them into a container containing the second liquid may be employed, similar to the nozzle part 32 described above. Alternatively, various known mixing means can be employed. Furthermore, as described above, an optical measurement container or the like may be used as the processing container for containing the second liquid.

[0094] In the optical measurement unit 7, the amount of chemiluminescence emitted from the second solution, which is a mixture of hydrogen peroxide solution and an alkaline solution, is detected by the second photodetector 71. The chemiluminescence detected here is, for example, a weak luminescence reaction with a measurement time of about 2 seconds. As the second photodetector 71, any known photodetector can be used, as long as it can amplify the signal as needed to detect the weak chemiluminescence, which is a minute amount of light emission, as the chemiluminescence amount. For example, a luminometer or a PMT (Photomultiplier Tube) can be used as the second photodetector 71.

[0095] In the optical measurement unit 7, when detecting the amount of light emitted from the chemiluminescent light-emitting part with the second light detection means 71, it is preferable to employ a light-shielding structure 73 to eliminate the influence of ambient light, since the chemiluminescence is a weak emission. The light-shielding structure 73 contains the second liquid that causes the light-emitting part to chemiluminescent. The processing container (which may also be an optical measurement container) is housed inside together with the second light detection means 71 to block ambient light when detecting the amount of chemiluminescence. The light-shielding structure 73 can be a commonly used light-shielding container, such as a dark box.

[0096] In other words, the optical measuring unit 7 having the second light detection means 71 preferably includes a pre-luminescence mixing means 72 for mixing hydrogen peroxide solution and an alkaline solution with the second liquid in a processing container that contains the second liquid processed in the second liquid processing unit 6, and a light-shielding structure 73 that houses the processing container together with the second light detection means 71 to block external light from the surroundings. The mixing of the second liquid with hydrogen peroxide solution and the alkaline solution using the pre-luminescence mixing means 72 may also be performed inside the light-shielding structure 73. In this case, even if weak chemiluminescence starts quickly due to the mixing, it is preferable because it becomes easier to quickly and reliably detect the amount of light emitted by the second light detection means 71 inside the light-shielding structure 73.

[0097] As a result of performing the series of processes on the blood sample as described above, the detected data includes the detection result regarding the optical concentration measurement of leukocytes in the first solution by the first photodetector and the detection result regarding the optical amount of chemiluminescence from the luminescent part of the gene where the luminescent part is formed by the second photodetector. The measurement processing unit 8 calculates the characteristics of specific regions of the genes possessed by the leukocytes from these detection results by the first photodetector and the second photodetector.

[0098] From the detection results of the first photodetector, an electrical signal with an intensity corresponding to the light intensity corresponding to the concentration of white blood cells in the first solution can be obtained. To calculate the white blood cell concentration from the detection results of the first photodetector, for example, a liquid equivalent to the first solution containing a known concentration of white blood cells is prepared, and the concentration is changed. Electrical signals with an intensity corresponding to the light intensity corresponding to each concentration are detected, and a calibration curve for the white blood cell concentration is obtained from these detection results. Then, the detection results obtained by the first photodetector according to the white blood cell concentration of the first solution are compared with the calibration curve, and the white blood cell concentration can be calculated from the detection results.

[0099] From the detection results of the second photodetector, an electrical signal with an intensity corresponding to the amount of chemiluminescence emitted from the luminescent portion of a gene where a luminescent portion has formed at a specific site in the second liquid can be obtained. To calculate the amount of chemiluminescence emitted from the luminescent portion from the detection results of the second photodetector, for example, a liquid equivalent to the second liquid containing leukocytes with G-tail 92 DNA of a known length at a known concentration is prepared, and the length and concentration are changed, and an electrical signal with an intensity corresponding to the amount of luminescence corresponding to each length and concentration is detected, and a calibration curve for the length of the G-tail 92 and the concentration of the leukocytes is obtained from these detection results. Then, the result calculated for the concentration of leukocytes in the first liquid is compared with the detection results of the second photodetector and the calibration curve, and the length of the G-tail 92 is calculated from the detection results. From this calculation result, the average length of the G-tail 92 obtained by comparing with the calibration curve can be determined.

[0100] Such a measurement processing unit 8 can use various known arithmetic processing means, computers, etc., and can execute processing programs, control programs, etc., according to the purpose of the measurement processing unit 8, and can be equipped with memory for calibration curves and detection results, etc., so various means, mechanisms, and devices can be used. Furthermore, there are no particular restrictions on the placement of the measurement processing unit 8 in the gene testing device 1, and as long as it can acquire detection results, exchange various signals for controlling each part, and display calculation results and control status to the user of the gene testing device 1, it may be arranged integrally with each part as a whole, or it may be arranged separately while maintaining the necessary connections to each part.

[0101] Furthermore, the measurement processing unit 8 not only processes the detection results from the first photodetector and the second photodetector, but also processes the sample container placement unit 2, the leukocyte separation unit 3, the concentration measurement unit 4, and reagents. The control unit may also function as a control unit that detects the operating status of each part, the mixing unit 5, the second liquid processing unit 6, and the optical measurement unit 7, and controls them appropriately. This would allow for automatic control of a series of processing operations in the gene testing device 1, enabling stable and efficient testing.

[0102] The above explanation mainly described the case where the specific site of a gene is the G-tail 92 of telomere 91, and the characteristic feature of the specific site of the gene is the length of the G-tail 92, i.e., the G-tail length. However, even when the specific site of a gene is telomere 91, and the characteristic feature of the specific site of the gene is the length of telomere 91, i.e., the telomere length, it can also be optically inspected using the gene testing device 1 of this disclosure. In that case, a label probe complementary to the telomere length of telomere 91 should be used. Whether the specific site of a gene is telomere 91 or the G-tail 92 of telomere 91, the probe reagent is the same telomere probe reagent. In either case, the processing procedure in the gene testing device 1 is the same as described above.

[0103] Although embodiments of this disclosure have been described in detail above, this disclosure is not limited to the embodiments described above, and various modifications and improvements are possible without departing from the gist of this disclosure. It goes without saying that all or part of each of the above embodiments and various modifications can be combined as appropriate and in a non-contradictory manner. [Explanation of Symbols]

[0104] 1. Genetic testing device 2. Sample container placement section 3 Leukocyte separation section 4 Concentration measuring section 5 Reagent mixing section 6. Second Liquid Treatment Process 7 Optical measurement section 8 Measurement Processing Unit 31. Leukocyte Separation Device 32 Nozzle section 33 Mobile drive unit 61 Heating means 62 At least one of heat dissipation means and cooling means 63 Temperature detection means 71 Second light detection means 72 Pre-emission mixing means 73 Light shielding structure 330 Flow channel section 331 Branch channel 332 holes 334 Main channel 3325 1st introduction hole (blood introduction hole) 3326 2nd discharge hole (red blood cell discharge hole) 3327 2nd introduction hole (liquid introduction hole for pressing) 3328 3rd drain hole (residual liquid drain hole) 3329 First discharge hole (white blood cell discharge hole) P100 Particles to be separated (white blood cells) P200 Other species particles (red blood cells)

Claims

1. A gene testing device that optically examines specific regions of genes present in white blood cells contained in the blood, A specimen container placement unit for placing specimen containers containing blood obtained by blood collection, and a leukocyte separation unit for collecting blood from the specimen containers in the specimen container placement unit, then separating leukocytes from the blood using a leukocyte separation device, and recovering a first solution containing leukocytes as the main blood cells. A concentration measuring unit comprising a first light incident means and a first light detection means for optically measuring the concentration of leukocytes in the first liquid, A reagent mixing unit prepares a second solution by mixing the cell membrane lysate with the first solution, adjusting the temperature, and then mixing in a probe reagent. A second liquid processing unit that heats and cools the second liquid to react a specific site of a gene in a leukocyte with the probe reagent, thereby forming a light-emitting portion at the specific site of the gene in the leukocyte in the second liquid, An optical measuring unit that mixes hydrogen peroxide solution and an alkaline solution with the second liquid containing the gene on which the light-emitting part is formed to cause the light-emitting part to chemiluminescent, and detects the amount of light emitted from the light-emitting part with a second photodetector, A measurement processing unit calculates the characteristics of a specific region of a gene possessed by a white blood cell from the detection results obtained by the first light detection means and the detection results obtained by the second light detection means. A genetic testing device equipped with the following features.

2. The leukocyte separation device is It comprises a plate-shaped base, a flow channel located inside the base, and a plurality of holes connected to the flow channel and opening on the outer surface of the base. The flow channel section includes a linear main flow channel extending in a first direction, and a plurality of linear branch flow channels connected to the first side surface of the main flow channel and narrower than the main flow channel, extending in a second direction perpendicular to the first direction. The plurality of holes include a first inlet for introducing blood, which is connected to the upstream part of the main flow path via a flow path extending in a first direction; a second inlet for introducing a pressing fluid, which is connected via a flow path to a second side surface opposite to the first side surface of the upstream part of the main flow path; a first discharge hole, which is connected to the first side surface of the downstream part of the main flow path via a flow path extending in a second direction; a second discharge hole, which is connected to the part of each of the plurality of branched flow paths opposite to the main flow path; and a third discharge hole, which is connected to the downstream part of the main flow path via a flow path extending in a first direction. The gene testing apparatus according to claim 1.

3. The leukocyte separation unit includes a nozzle unit having a syringe pump function for aspirating blood from the sample container and injecting the blood into the leukocyte separation device, and a moving drive unit that moves the nozzle unit from the sample container to the leukocyte separation device for blood aspiration and injection. The gene testing apparatus according to claim 1.

4. The reagent mixing unit includes a lysis solution mixing means for mixing the cell membrane lysis solution, a temperature adjustment means for heating and cooling the mixed solution after mixing the cell membrane lysis solution with the first solution, and a reagent mixing means for mixing the probe reagent. The gene testing apparatus according to claim 1.

5. The second liquid processing unit comprises a processing container for containing the second liquid, a heating means for heating the processing container, at least one of a heat dissipation means and a cooling means for cooling the processing container, and the processing container It has a temperature detection means for detecting the temperature of the container, The gene testing apparatus according to claim 1.

6. The optical measuring unit includes a pre-light mixing means for mixing the hydrogen peroxide solution and the alkaline solution with the second liquid in a processing container that contains the second liquid processed in the second liquid processing unit, and a light-shielding structure that houses the processing container together with the second light detection means to block external light from the surroundings. The gene testing apparatus according to claim 1.

7. The aforementioned specific region is a telomere, and the characteristic feature of the aforementioned specific region is its telomere length. The gene testing apparatus according to claim 1.

8. The gene testing apparatus according to claim 1, wherein the specified region is the G-tail of a telomere, and the characteristic feature of the specified region is the length of the G-tail.

9. The probe reagent is a telomere probe reagent. The gene testing apparatus according to claim 6 or 7.

10. The sample container placement unit, the leukocyte separation unit, the concentration measurement unit, the reagent mixing unit, and the second liquid processing unit are arranged in this order in the left-right direction. The optical measuring unit is positioned rearward between the concentration measuring unit, the reagent mixing unit, and the second liquid processing unit. The gene testing apparatus according to claim 1.

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